\documentclass[a4paper]{article}

\usepackage[english]{babel}
\usepackage[utf8x]{inputenc}
\usepackage{amsmath}
\usepackage{float}
\usepackage{graphicx}
\usepackage[colorinlistoftodos]{todonotes}
\usepackage[authoryear]{natbib}
\usepackage{hyperref}

\title{Microbiological Water Safety Monitoring, Spring 2016}
\author{Jacqueline Dokko, Janak Shah}
\date{\today} % You can write in any date within the brackets.

\begin{document}

\maketitle
 % * <akl62@cornell.edu> 2016-02-17T04:26:11.657Z:
%
% An abstract on the title page would be good and then make the table of contents on it's own separate page. Resolved
%
% ^ <akl62@cornell.edu> 2016-02-24T05:17:45.540Z.
% * <akl62@cornell.edu> 2016-03-15T14:21:35.578Z:
%
% I don't know if you did this before the email was sent out, but instead of writing resolved on my comment, you should just reply with resolved and then I will close it
%
% ^ <jhs373@cornell.edu> 2016-04-07T19:54:55.202Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-04-26T16:17:04.908Z.
% * <akl62@cornell.edu> 2016-03-01T15:14:30.323Z:
%
% Is this an new document? If so, you should copy and paste the comments from the previous document into the same positions on this document. Also, I would label the other document as solely task list and lit review. Resolved
%
% ^ <akl62@cornell.edu> 2016-03-15T14:21:18.670Z.
\begin{abstract}
The ultimate goal for the microbiological water safety monitoring (MWSM) team is the development of a test that detects pathogens in water.  The test must be of low cost (under ten dollars for each test), have a reduced incubation time from the standard 48 hours, and be able to be used in a low-resource setting such as Honduras. The team tested methods indicating the  presence or absence of bacteria compared to quantitatively determining bacterial presence. Upon understanding the cost and efficiency of each method, it was possible to narrow down the methods that could be used as a model for microbial detection for AguaClara purposes. 
% * <cpf45@cornell.edu> 2016-04-11T18:50:49.940Z:
%
% > Considering the ultimate goals for the microbiological water safety monitoring team, it is necessary to research and understand the options available for pathogenic detection in water.
%
% You should specifically state what the 'ultimate goals' of this team are in the abstract (one sentence!).
%
% ^ <akl62@cornell.edu> 2016-05-14T16:35:53.353Z.
% * <akl62@cornell.edu> 2016-03-15T14:25:09.757Z:
%
% I would write in here some of the quantitative goals such as reducing cost,  reducing culture time, presence absence vs quantatative and such. 
%
% ^ <jhs373@cornell.edu> 2016-04-05T19:42:41.522Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-04-26T16:19:43.937Z.
%
% ^.
% * <akl62@cornell.edu> 2016-03-01T15:16:52.386Z:
%
% > AguaClara's purposes 
%
% AguaClara Resolved
%
%
% ^ <akl62@cornell.edu> 2016-03-15T14:25:05.792Z.
\end{abstract}
% * <akl62@cornell.edu> 2016-02-24T04:54:29.541Z:
%
% Make sure you make a new overleaf using the template Theresa sent out. All you have to do is just copy and paste the code for each section on this file onto the other one. Also make sure to update your task list with the comments I suggested. Resolved
%
% ^ <akl62@cornell.edu> 2016-03-15T14:26:56.894Z.
% * <akl62@cornell.edu> 2016-02-17T04:25:48.573Z:
%
% I would indent the subsections to make it look better.
%
% ^ <akl62@cornell.edu> 2016-03-15T14:27:19.649Z.
\section*{Introduction}
% * <cpf45@cornell.edu> 2016-04-14T15:38:11.216Z:
%
% > Introduction
%
% Overall, good intro to the work!
%
% ^ <akl62@cornell.edu> 2016-04-26T16:21:24.117Z.
The goal of this team was to develop a test that could be used to determine the safety of the water treated by the AguaClara plants by ensuring that there are no pathogens in this water. A sample of water can only be certified to be safe if there are no microbes detected. Any presence of microbes would allow for reproduction and population increase, and would cause dietary health issues for widespread consumers. Thus only a presence/absence test for microbes was all that was required, but the team hoped to design a quantification device such that the data collected could be used by plant operators to adjust filtration accordingly. Though the most recent Honduras team tested the water treated by a newly installed plant and found it to be free of E. coli, a number of events could take place during the lifetime of a plant which could affect its ability to clean water. A leak or failure of one of the pipes or tanks, improper operation and cleaning of parts of the tank including the backwashing of filters, and increased precipitation during the rainy season all affect the plant's effectiveness and would require the E. coli content of the treated water to be re-evaluated. Typical microbiological water tests are often expensive and must be carried out in a lab. However, conditions at the plant sites made these kinds of tests impractical. Additionally, in order to be certain that AguaClara plants successfully eliminate pathogens from the water, microbiological tests on the treated water must be run once a day. The method previously used cost 10 dollars per test, which put a significant financial strain on the communities in which these plants operate. For these reasons, the MWSM team was created to develop a cheap, fast, and reliable test that can be used in a low-resource setting and to produce results which can be interpreted by plant operators. This new test would not only allow the operators to easily and inexpensively confirm the safety of the treated water before it is sent out of the plant, but would also provide valuable information on which processes in the plant are eliminating microbial contamination.
% * <cpf45@cornell.edu> 2016-04-11T20:56:51.435Z:
%
% and reliable?
%
% ^ <jmd475@cornell.edu> 2016-04-22T13:51:32.084Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:56.526Z.
% * <cpf45@cornell.edu> 2016-04-11T18:56:25.986Z:
%
% > MWSM
%
% Define this acronym before using it!
%
% ^ <jmd475@cornell.edu> 2016-04-22T13:51:40.887Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:49.933Z.
% * <cpf45@cornell.edu> 2016-04-11T18:54:34.435Z:
%
% > determine the safety of the water treated by the AguaClara plants
%
% How are you determining safety? I would elaborate here before jumping into what was seen in Honduras.
%
% ^ <jmd475@cornell.edu> 2016-04-22T13:51:46.976Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:51.778Z.
% * <akl62@cornell.edu> 2016-03-15T14:30:30.443Z:
%
% > lab,
%
% lab.
%
% ^ <jhs373@cornell.edu> 2016-04-05T19:43:59.102Z: Resolved
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:53.692Z.
%
% ^.
% * <akl62@cornell.edu> 2016-03-15T14:29:29.730Z:
%
% >  backwashing filters
%
% the backwashing of the filters
%
% ^ <jhs373@cornell.edu> 2016-04-05T19:51:52.615Z:
%
% Resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:46.959Z.
% * <akl62@cornell.edu> 2016-03-15T14:28:14.401Z:
%
% Make sure you write in past tense for this intro paragraph. It seems like it's mostly in the present. Things that will always be here should be in present but anytime you are talking about something you did it should be in the past.
%
% ^ <jhs373@cornell.edu> 2016-04-07T19:54:25.152Z:
%
% Resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:45.573Z.
% * <akl62@cornell.edu> 2016-03-01T15:23:20.160Z:
%
% > improper operation causing the filters to not be cleaned often enough
%
% improper operation and cleaning of parts of the tank including bakwashing filters,
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:29:19.961Z.
% * <akl62@cornell.edu> 2016-03-01T15:22:15.000Z:
%
% > of events take
%
% of events could
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:33:35.744Z.

\section*{Literature Review}

\subsection*{\textit{Low Resource Setting Experimentation}}
% * <akl62@cornell.edu> 2016-03-01T15:26:44.450Z:
%
% > Low Resource Setting Experimentation}
%Resolved
% I would italicize topic headings
%
% ^ <akl62@cornell.edu> 2016-03-15T14:32:58.655Z.
Murcott et al.  (2015) was a paper on the effectiveness of different detection methods in areas with low resources such as Ghana and India. The "effectiveness" was a culmination of the quantified records for accuracy, precision and speed. This paper's locations and its resources are parallel to those of the water treatment plants of AguaClara. The study was conducted by comparing the effectiveness of the detector using ground water of unknown bacterial concentration and bottled water of known bacterial concentration. Overall, the two different settings of India and Ghana were shown to have slightly differing results but nothing of significance. For some reason, some of the tests fared better in Ghana than in India based on Total Coliform Performance Ratings which was basically a method for comparing tests that used consumer reports as the main source of information. The methods tested were: Hydrogen Sulfide TARA Aquacheck, ORlab H2S, Lab made H2S test, IDEXX Quantitray 2000, 3M Petrifilm, EasyGel cards, Aquagenx compartment bags \cite{murcott_evaluation_2015}. The TARA Aquacheck and ORlab both determined the quantity of bacteria by picking up traces of hydrogen sulfide which were produced by the target bacteria. The Lab made H2S test was a more accurate version of TARA and ORlab, but requires a laboratory setting. The IDEXX Quantitray 2000 used a dish with many chambers to statistically approximate the quantity of microbes but requires a laboratory setting as well. The statistical method commonly used called the most probable number method compares the number of innoculated chambers against the number of total chambers to calculate the approximate original microbe concentration in the water. The 3M Petrifilm used a color changing chemical indicator and a small amount of sample water which lead to inaccurate quantification readings. The EasyGel cards was a scaled down version of the Quantitray that only required 1mL of sample but only provided inaccurate readings. The Aquagenx compartment bags used the same concept as the Quantitray but with different sized compartments of bags and without the requirement of a laboratory setting.  These tests were a mix of quantifiable detection and presence-absence detection methods. The results took into consideration the cost, specificity, and sensitivity, thus addressing key problems in developing a detector: applicability, accuracy and precision. In terms of reliability and accuracy, the three top forms of detection were IDEXX Aquacheck, ORLab, and lab-made H2S tests , which all required high technology lab settings thus were determined to be infeasible for the situations in the AguaClara water plants. 

When testing the methods, the researchers looked for low false positives and low false negatives which are both important in determining the true quality of the water. A false positive occurs when a test incorrectly indicates the presence of a condition, while a false negative occurs when a test incorrectly indicates the absence of a condition. In this system, a false positive would occur when water that is pathogen-free is labeled as contaminated. This could lead to an increase in filtration mechanisms which would unnecessarily increase costs. On the other hand, false negatives are also harmful for treatment plants because that would mean that contaminated water would be passed off as safe water, thus putting the consumers at risk. False positives and false negatives must both be minimized to ensure the reliability of tests.
% * <akl62@cornell.edu> 2016-04-26T16:26:04.558Z:
%
% > a false negative would occur when water that is contaminated with pathogens is incorrectly identified as pathogen free. This could lead to an increase in filtration mechanisms which would unnecessarily increase costs. On the other hand, false negatives are also harmful for treatment plants because that would mean that contaminated water would be passed off as safe water, thus putting the consumers at risk.
%
% You mention false negative twice. I think you should change the first one to false positive since you talk about increasing costs right after
%
% ^ <jhs373@cornell.edu> 2016-05-06T18:47:45.720Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:37:58.906Z.

Considering the cost, sensitivity and specificity, the compartment bags proved to be most realistic. Although the compartment bags only indicate the presence or absence of bacteria, the water plants seem to only require that much, thus the compartment bags may be useful for AguaClara. However, the compartmental bags require too much incubation time to provide useful information for plant operators in the time required. In the case that this sort of delay is acceptable, the compartmental bag test seemed to be the most viable in low resource settings.\cite{murcott_evaluation_2015}.
% * <cpf45@cornell.edu> 2016-04-13T15:29:32.442Z:
%
% You should start a new paragraph here.  Define false negative and false positive and then use them in context. For instance: a false negative occurs when a test incorrectly indicates the absence of a condition. In these systems, a false negative occurs when water that is contaminated with pathogens is incorrectly identified as being pathogen-free (safe, etc)... and continue to explain the effects of releasing contaminated water to the public. 
%
% ^ <jmd475@cornell.edu> 2016-04-22T13:59:43.834Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:37.285Z.
% * <cpf45@cornell.edu> 2016-04-13T15:28:56.492Z:
%
% > putting the consumers at bay
%
% You mean at risk??
%
% ^ <jmd475@cornell.edu> 2016-04-22T13:59:48.895Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:34.739Z.
% * <cpf45@cornell.edu> 2016-04-13T15:13:39.667Z:
%
% > These tests were a mix of quantifiable detection and presence-absence detection methods.
%
% It would be infromative to the reader to know which ones were quantitative and which were presence absence particularly when you mentions the top 3 technologies later on.
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:14:32.468Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:32.739Z.
% * <cpf45@cornell.edu> 2016-04-13T15:09:02.356Z:
%
% > The methods tested were: Hydrogen Sulfide TARA Aquacheck, ORlab H2S, Lab made H2S test, IDEXX Quantitray 2000, 3M Petrifilm, EasyGel cards, Aquagenx compartment bags 
%
% You should describe these tests so the reader knows what you're talking about! A brief summary of each type of test would be good.
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:12:27.236Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:29.804Z.
% * <cpf45@cornell.edu> 2016-04-13T15:08:19.843Z:
%
% > Overall, the two different settings of India and Ghana were shown to have slightly differing results but nothing of significance
%
% What were the results?
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:28:52.741Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:27.821Z.
% * <cpf45@cornell.edu> 2016-04-11T21:05:26.382Z:
%
% > Considering how recent the paper was published, it was hard to determine the reliability; however, it also indicated that the methods listed were up-to-date.
%
% What do you mean by this?
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:29:01.835Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:25.020Z.
% * <cpf45@cornell.edu> 2016-04-11T21:02:30.357Z:
%
% > effectiveness
%
% what do you mean by the effectiveness? How is this measured?
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:29:09.334Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:23.442Z.
% * <cpf45@cornell.edu> 2016-04-11T21:00:05.290Z:
%
% > Amongst the articles researched was a paper on the effectiveness of different detection methods in areas with low resources such as Ghana and India. This paper's locations and its resources are parallel to those of the water treatment plants of AguaClara. 
%
% If you are talking about a specific study you should cite it. I would reword this and include a citation (e.g. Smith et al (2016) studied the effectiveness of different detection methods...)
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:14.645Z:
%
% resolved
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:31:17.602Z.
% * <akl62@cornell.edu> 2016-02-24T04:52:45.407Z:
%
% I recommend that you split the lit review into a couple main topics and then give bolded labels that show what the following paragraphs will talk about
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:34:21.879Z.
% * <akl62@cornell.edu> 2016-03-01T15:33:20.027Z:
%
% > ere up-to-date. These tests were a mix of quantifiable detection and presence-absence detection methods. The results took into consideration the cost, specificity, and sensitivity, thus addressing key problems in developing a detector: applicability, accuracy and precision. The three top forms of detection were Aquacheck, ORLab, and H2S tests, which all required high technology lab settings which are not feasible for the situations in the AguaClara water plants. When testing the methods, the researchers looked for low false positives and low false negatives which are important in determining the true quality of water. Considering the cost, sensitivity and specificity, the compartment bags proved to be most realistic. Although the compartment bags only indicate the presence or absence of bacteria, the water plants seem to only require that much, thus the compartment bags may be useful for AguaClara. However, considering the future goal of a sliding colony counter, the compartmental bags require too much incubation time for it to provide useful information for plant operators in the time required. In the case that this sor
%Resolved
% Is any of this part paraphrase? If so, I would add another citation near the bottom of the paragraph
%
% ^ <akl62@cornell.edu> 2016-03-15T14:37:23.027Z.
% * <akl62@cornell.edu> 2016-03-01T15:31:41.671Z:
%
% >  low false positives and low false negatives
%Resolved
% I would expand on this for people who read this but don't have prior knowledge on these terms
%
% ^ <akl62@cornell.edu> 2016-03-15T14:37:31.790Z.
% * <akl62@cornell.edu> 2016-02-24T05:02:12.666Z:
%
% Make sure you write in past tense because all the papers you read are conducted research in the past.
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:37:33.611Z.


\subsection*{\textit{Summary of Different Tests}}
% * <cpf45@cornell.edu> 2016-04-13T15:55:16.541Z:
%
% > Summary of Different Tests
%
% This section was hard to follow. Were they actually testing these kits in different settings? If so did they say which ones worked best? Or were they just looking at tests already being used in different resource settings?
%
% ^ <jhs373@cornell.edu> 2016-04-22T23:40:18.654Z:
%
% resolved through table of results
%
% ^ <akl62@cornell.edu> 2016-05-03T15:27:40.107Z.
A study was done by several institutions that summarized different microbial drinking water tests.\cite{bain_summary_2012} This study identified 44 different tests, with 18 being presence/absence tests, and the other 26 quantitative tests. The goals of this study were similar to the goals of this subteam: they defined primary characteristics to be considered when selecting a test for drinking water and combined this information to find a suitable test given the resources available. Three different resource settings were tested: low resource, medium resource, and high resource. In a low resource setting, there is no laboratory or electricity by the water source. A medium resource setting is one with a basic laboratory and electricity within 24 hours of the laboratory. A high resource setting is one with a regular laboratory and running electricity. The laboratory in this setting is comparable to standard university testing labs \cite{bain_summary_2012}. The low resource setting is the most comparable setting to the target site in Honduras. The tests were then evaluated for different conditions. The conditions that determined how the tests were compiled included cost, detection of faecal indicator bacteria, development of the test, and whether there was at least 1 ml volume of the sample \cite{bain_summary_2012}. Most tests were relatively cheap, and as predicted: the tests that utilized E-coli as the indicator organism tended to be more prevalent in a low-resource setting.\cite{bain_summary_2012}. Tests using total coliforms and hydrogen sulfide rather than E-coli were also included. \cite{bain_summary_2012}. This study is valuable because it compiles every test and aids in the selection process. 
% * <akl62@cornell.edu> 2016-05-14T16:39:29.672Z:
%
% > This is the most comparable setting to the target site in Honduras.
%
% This makes it sound like the high resources setting is the most comparable when the low resource setting is most comparable. I would specifically state that and fix this
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:38:40.767Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-19T19:53:21.177Z.
% * <akl62@cornell.edu> 2016-04-28T14:38:04.715Z:
%
% >  medium resource, and high resource
%
% I would define what these setting are too.
%
% ^ <jhs373@cornell.edu> 2016-05-05T20:36:30.401Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:39:08.431Z.
% * <akl62@cornell.edu> 2016-04-28T14:25:31.621Z:
%
% > summarizes
%
% summarized
%
% ^ <jhs373@cornell.edu> 2016-05-05T20:37:03.079Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:39:14.139Z.
The table below shows the different presence/absence tests, most probable number tests, which are estimates of the amount of pathogens, and different enumeration tests. The figure evaluates price, incubation time, and other factors. 
% * <akl62@cornell.edu> 2016-04-28T14:33:26.601Z:
%
% > The table below shows the different presence/absence tests as well as most probable number tests, which are estimates of the amount of pathogens. The figure evaluates price, incubation time, and other factors. 
%
% These two images are difficult to see because the writing is so small. Is there anyway to make writing bigger? Or increase the resolution?
%
% ^ <jmd475@cornell.edu> 2016-05-18T16:12:15.601Z:
%
% resolved?
%
% ^ <akl62@cornell.edu> 2016-05-19T19:57:02.061Z.
\begin{figure}[H]
\centering
\includegraphics[scale=0.30]{Tests.png}
\caption{Table of Tests}
\label{Design goal}
\end{figure}

% * <cpf45@cornell.edu> 2016-04-13T15:56:57.664Z:
%
% > The tests were relatively cheap, and as predicted: the tests that utilized E-coli as the indicator organism tended to be more prevalent in a low-resource setting.\cite{bain_summary_2012}. Tests using total coliforms and hydrogen sulfide were also included. Most tests had an incubation time of 24 hours as well, which was expected\cite{bain_summary_2012}. This study is valuable because it compiles every test and aids in the selection process. 
%
% You should explain what these different types of tests entail, which are quantitative which are presence-absence. A table showing comparisons of the different tests would be great here!
%
% ^ <jhs373@cornell.edu> 2016-04-22T23:40:23.894Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:27:52.357Z.
% * <akl62@cornell.edu> 2016-02-24T05:06:29.727Z:
%
% When doing text citations, make sure you put the period after the citation.
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:38:15.087Z.
% * <akl62@cornell.edu> 2016-03-01T15:35:28.466Z:
%
% > whether the test was developed or not
%
% rephrase, maybe development of the test
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:38:59.498Z.
% * <akl62@cornell.edu> 2016-03-01T15:34:40.514Z:
%
% > ed, 
%
% Change the comma to a :
%
% ^ <jhs373@cornell.edu> 2016-04-05T20:02:31.356Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-04-28T14:28:23.059Z.

\subsection*{\textit{Compartmentalized Bag Test}}
A study was performed specifically on the compartmentalized bag test, which is one of the more developed and well-known tests, that tested its accuracy by comparing it to a standard lab filtration test using agar.\cite{stauber_evaluation_2014}. The filtration test used a selective medium containing chromogenic and fluorogenic Beta-glucuronide and Beta-galactoside substrates used for the detection and quantification of E. coli and coliforms. Chromogenic substrates react with proteolytic enzymes, or enzymes that break down proteins, to create the color change that identifies E-coli and coliforms. Fluorogenic substrates, however, react with enzymes to produce a fluorescent compound, which also identifies E-coli and coliforms\cite{stauber_evaluation_2014}. The bag test, however, was based on the metabolism of glucuronides to the enzyme Beta-glucuronidase. This reaction caused the color change to blue, as shown in the figure below.  This blue color indicated the presence of E-coli\cite{stauber_evaluation_2014}. The two different tests agreed on results 70, 71.4, 73.5, and 92.8 percent of the time for four E. coli concentrations of \textless1, 1–10,11–100, and \textgreater100 MPN/100 ml respectively  \cite{stauber_evaluation_2014}. The higher concentrations of E-coli returned the more accurate results. There were some problems found with the compartmentalized bag test, including false negative readings. When the test indicated the presence of pathogens, it was 95 percent accurate, but if it returned a negative there was a  chance that the water was still contaminated, which is an example of the previously defined false negative result to a test. Out of 263 samples tested with both the compartmentalized bag test and the membrane filtration test, 12 were found to be negative for pathogen presence with the bag test but positive for pathogen presence with the membrane filtration test. Therefore, there was a 4.6 percent chance that a false negative test occurred in this experiment \cite{stauber_evaluation_2014}. Overall, however, the compartmentalized bag test was found to be generally reliable and an efficient test in that it can be used when other microbial water testing resources are not available.
% * <akl62@cornell.edu> 2016-05-03T14:41:57.321Z:
%
% > the compartmentalized bag test, which is one of the more developed and well-known tests, that tested its accuracy by comparing it to a standard lab filtration test using agar.
%
% Try to get any image of a compartment bag. and possibly of the filtration test
%
% ^ <jhs373@cornell.edu> 2016-05-06T18:52:45.092Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:44:02.937Z:
%
% Do you have a picture of the bag without any fluid in it showing all the different sized comparments?
%
% ^ <jhs373@cornell.edu> 2016-05-16T00:24:30.616Z:
%
% Resolved
%
% ^.
% * <akl62@cornell.edu> 2016-05-03T14:40:57.466Z:
%
% > These substrates reacted
%
% Is there any way you can be more specific about this?
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:38:01.341Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:46:41.630Z.
% * <akl62@cornell.edu> 2016-05-03T14:38:43.315Z:
%
% > This is an example of a false negative result to a test. 
%
% I would try to work this into the previous statement and not just add a new sentence.
%
% ^ <jhs373@cornell.edu> 2016-05-05T20:43:10.505Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:46:45.043Z.
% * <akl62@cornell.edu> 2016-05-03T14:37:41.216Z:
%
% > higher chance that the water was still contaminated
%
% Cristina I think meant % chance. So do you have a number for this?
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:29:16.585Z:
%
% Resolved
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:29:26.056Z:
%
% Changed the wording as well to make it more coherent
%
% ^ <akl62@cornell.edu> 2016-05-14T16:46:54.247Z.
% * <akl62@cornell.edu> 2016-04-28T14:47:05.816Z:
%
% >  color change
%
% Mention from what color to what color shows presence. Or just comment that blue or yellow is the contaminated compartment in the figure
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:30:00.280Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:47:09.715Z.
% * <akl62@cornell.edu> 2016-04-28T14:44:09.059Z:
%
% > These substrates react and  
%
% Did you forget to finish this?
%
% ^ <jhs373@cornell.edu> 2016-04-28T19:12:15.012Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:39:14.168Z.
% * <cpf45@cornell.edu> 2016-04-13T17:10:38.090Z:
%
% >  while if it returned a negative there is a relatively higher chance that the water was still contaminated 
%
% A great place to mention false negatives!
%
% ^ <cpf45@cornell.edu> 2016-04-13T17:11:23.722Z:
%
% Also what is the %change that the water is still contaminated?
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:58:07.627Z:
%
% resolved
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:58:30.912Z:
%
% report does not say %chance
%
% ^ <akl62@cornell.edu> 2016-05-03T14:39:21.903Z.
% * <cpf45@cornell.edu> 2016-04-13T17:08:08.680Z:
%
% > The filtration test used a selective medium containing chromogenic and fluorogenic Beta-glucuronide and Beta-galactoside substrates for the detection and quantification of E. coli and coliforms \cite{stauber_evaluation_2014}. The bag test, however, was based on the metabolism of glucuronides to Beta-glucuronidase \cite{stauber_evaluation_2014}.
%
% These methods should be explained more in detail.  How do the different substrates help quantify e coli? A reader that doesn't know these tests would have no idea what you're talking about! A graphic might be helpful here
%
% ^ <jhs373@cornell.edu> 2016-04-22T23:53:58.574Z:
%
% I can't find a graphic depicting this, but clarified in the comments
%
% ^ <akl62@cornell.edu> 2016-05-14T16:47:32.363Z.
% * <cpf45@cornell.edu> 2016-04-13T17:06:08.937Z:
%
% > <
%
% Proofread your report, the < and > show up as different symbols in the actual document
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:53:56.364Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-04-28T14:45:42.903Z:
%
% These things still show up as different symbols.
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:39:58.342Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:47:38.144Z.
% * <cpf45@cornell.edu> 2016-04-13T16:04:39.720Z:
%
% > by Christine Stauber, Candace Miller, Brittany Cantrell, and Kate Kroell.
%
% Work on using APA citation style.
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:53:52.844Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:43:05.178Z.

\begin{figure}[H]
\centering
\includegraphics[scale=0.35]{bagtest.JPG}
\caption{Color change reflects presence of E-coli}
\label{Compartmentalized Bag Test}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.45]{membrane_filtration.jpg}
\caption{The standard membrane filtration test}
\label{Filtration Test}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.5]{compartments.JPG}
\caption{The different size compartments are shown in this figure.The specified volume of each compartment is what is put into each.}
\label{Empty Compartments}
\end{figure}
% * <akl62@cornell.edu> 2016-03-15T14:42:57.562Z:
%
% > l
%
% 100 ml respectively.
%
% ^ <jhs373@cornell.edu> 2016-04-05T20:03:10.490Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-04-28T14:29:55.800Z.
% * <akl62@cornell.edu> 2016-03-15T14:50:50.495Z:
%
% Is it possible to resuse this bags and if you can't, why not? Also is it possible to perform these tests in ziploc bags using different amount of water just like the different compartments? Just an idea
%
% ^.
% * <akl62@cornell.edu> 2016-02-24T05:10:00.105Z:
%
% >  The tests were found to agree on results 70, 71.4, 73.5, and 92.8 percent of the time for E. coli concentration categories of < 1, 1–10,11–100, and >100 MPN/100 ml or CFU/100 ml, respectively.
%
% This part is a little confusing. You should try to reword it and make it more concise.
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:42:15.804Z.
% * <akl62@cornell.edu> 2016-03-01T15:38:22.224Z:
%
% > four different E. coli concentrations 
%
% I would list the E. coli concentrations if you know them, or say with increasing concentrations of E.coli
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:42:36.071Z.

\subsection*{\textit{MBS Method}}
In an effort to develop a test for contaminants in drinking water which did not require a laboratory, expensive equipment, or trained technicians, researchers developed the Microbiological Survey Method. This method, which makes use of total coliforms as an indicator of contamination, uses disposable reaction vials that "measure the catalytic activity of redox enzymes of the main metabolic pathways of bacteria" and can be read by a change in color of the contents of the vial \cite{arienzo_field_2015}. To test this method, sixty-one wells were made around Douala, Cameroon. The majority of these wells were dug wells between 1.5m and 6m in depth, while others were drilled wells deeper than 20m. Water samples from each well were collected in each of three distinct seasons: once in the rainy season characterized by heavy, frequent rainfall and temperatures around 30-38 degrees Celsius, once in the dry season with no rainfall and high temperatures around 40-45 degrees Celsius, and once in the beginning of the rainy season, which has both high temperatures and heavy precipitation. These water samples came from climates similar to those of Honduras though from differing sources (well water from Douala versus surface water in Honduras), and water from both Honduras and Douala were often contaminated with faecal bacteria. The rainy season facilitated the growth of more microbes and the drilled wells tended to have more contaminated samples. The difference in water sources between the Honduran and Cameroon water would have been the cause for incomparable results since surface water and well water are two different environments. For this method, the quantity of CFUs (Colony Forming Units) were used to standardize and compare the results of each experiment. Researchers commonly refer to colonies as CFUs as a way of assuming that one colony grew from only one cell for the sake of simplicity.
% * <akl62@cornell.edu> 2016-05-14T16:48:55.200Z:
%
% > These water samples came from environments similar to those found in Honduras
%
% You still need to clarify how this is similar to Honduras, because Honduras uses surface water while it seems the data you put on here is from ground, or well, water, which would not be comparable
%
% ^ <akl62@cornell.edu> 2016-05-19T20:02:30.496Z.
% * <akl62@cornell.edu> 2016-05-03T14:49:24.373Z:
%
% > n cause
%
% been the cause?
%
% ^ <jhs373@cornell.edu> 2016-05-05T20:44:25.686Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:47:54.964Z.
% * <akl62@cornell.edu> 2016-05-03T14:49:07.492Z:
%
% >  source
%
% sources
%
% ^ <jhs373@cornell.edu> 2016-05-05T20:44:34.645Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:47:59.067Z.
% * <akl62@cornell.edu> 2016-05-03T14:48:01.190Z:
%
% > The majority of these wells were dug wells between 1.5m and 6m in depth, while others were drilled wells deeper than 20m. Water samples from each well were collected in each of three distinct seasons
%
% You say here that water was collected from the wells, but later on you say its obtained from surface water sources. Which one is it?
%
% ^ <akl62@cornell.edu> 2016-05-03T14:51:01.112Z:
%
% you also say that it has comparable environments, but there are inaccurate results, so then why compare it to Honduras if you know that there will be inaccurate results?
%
% ^ <akl62@cornell.edu> 2016-05-19T20:02:35.416Z.
% * <cpf45@cornell.edu> 2016-04-13T18:23:57.011Z:
%
% > These water samples came from environments similar to those found in Honduras, as water from both Honduras and Douala is often contaminated with faecal bacteria and is obtained from surface water sources, meaning varying amounts of precipitation can affect its safety as drinking water. 
%
% This would be a good place to summarize the results they found by season. I'm assuming they saw higher total coliforms in the rainy season and less in the dry season?
%
% ^ <jmd475@cornell.edu> 2016-04-22T20:01:17.646Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:47:38.331Z.
% * <cpf45@cornell.edu> 2016-04-13T18:17:13.503Z:
%
% > surface water sources
%
% From what you wrote, my understanding is that the water samples were taken from wells not surface waters so are they really comparable to Honduras?
%
% ^ <jmd475@cornell.edu> 2016-04-22T19:55:01.985Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-19T20:02:37.744Z.
% * <cpf45@cornell.edu> 2016-04-13T18:04:07.888Z:
%
% > three distinct seasons
%
% Are the 'distinct' seasons: rainy season, dry season, and beginning of rainy season? 
%
% ^ <jmd475@cornell.edu> 2016-04-22T19:53:15.143Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:48:33.613Z.
% * <cpf45@cornell.edu> 2016-04-13T17:14:14.456Z:
%
% > This method, which makes use of total coliforms as an indicator of contamination, uses disposable reaction vials that "measure the catalytic activity of redox enzymes of the main metabolic pathways of bacteria" and can be read by a change in color of the contents of the via
%
% This is a great, short description of the method. You should include the same for all methods you mention.
%
% ^ <akl62@cornell.edu> 2016-05-03T14:48:44.965Z.

To develop a timeline for the color change of the vial content, water samples with known amounts of contaminant were tested in the lab. 
The water samples from Cameroon were treated with thiosulfate to inhibit the antimicrobial effects of the chlorine that was being used to treat the water. The results of the test are interpreted through the change in color of the vial content over time: color change after 14 hours indicates a level of total coliforms above 100 CFU/mL, color change only after 24 hours indicated total coliforms lower than 100 CFU/mL, and no color change indicates the absence of any coliforms. A visual of the color change can be seen in figure \ref{MBS}
% * <akl62@cornell.edu> 2016-05-03T14:52:06.752Z:
%
% > gh the change in color of the vial content o
%
% Do you have a picture of this method or showing color change?
%
% ^ <akl62@cornell.edu> 2016-05-14T16:50:47.988Z:
%
% Put a picture of a file here if you find one
%
% ^ <akl62@cornell.edu> 2016-05-19T20:02:57.026Z.
% * <cpf45@cornell.edu> 2016-04-13T18:34:57.714Z:
%
% > CFU
%
% Define CFUs somewhere...
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:44:50.738Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:51:36.194Z.

\begin{figure}[H]
\centering
\includegraphics[scale=0.7]{MBS.jpg}
\caption{The color change used for the microbiologicla survey method.}
\label{MBS}
\end{figure}

The MBS method, when tested on the water samples from Cameroon, was found to underestimate the bacterial concentration by 0.5 log units, or units defined on a logarithmic scale, whereas the most probable number technique has a given uncertainty of about 1 log unit. The most probable number technique works by identifying the most likely number of pathogens in a sample. However, this only applies when the test is performed in the presence of a coliform concentration of 100 CFU/mL or higher. When the bacterial concentration (expressed as the log of CFU/mL) was plotted against water sample color change time, a high level of correlation (0.98) was found in the relationship between the MBS method and the bacterial concentration. The correlation factor between the CFU/mL obtained via traditional methods and the MBS method was 0.94 for total coliforms \cite{arienzo_field_2015}. Although the water sources of Douala and Honduras are different, and this method may not be completely applicable to the MWSM team, the team was made aware of this method and the logic of this method could contribute to the development of AguaClara's microbe detector. 
% * <akl62@cornell.edu> 2016-05-03T14:53:35.754Z:
%
% > this only 
%
% Is this most probable number technique? Also did you define what the technique was?
%
% ^ <akl62@cornell.edu> 2016-05-14T16:51:53.721Z.
% * <cpf45@cornell.edu> 2016-04-13T21:06:08.585Z:
%
% > When the bacterial concentration (expressed as the log of CFU/mL) was plotted against the time occurring for color change of the water samples, a high level of correlation (0.98) was found to the linear inverse relationship between the MBS method and the bacterial concentration.
%
% This sentence is confusing, I think I understand what you're saying but it should be reworded.
%
% ^ <jmd475@cornell.edu> 2016-04-22T19:40:58.751Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:56:24.079Z.
% * <cpf45@cornell.edu> 2016-04-13T18:41:25.830Z:
%
% > 100
%
% units?
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:45:36.927Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:52:58.260Z.
% * <cpf45@cornell.edu> 2016-04-13T18:33:46.630Z:
%
% > whereas the most probable number technique has a given uncertainty of about 1 log unit
%
% Did you define this method anywhere?
%
% ^ <jmd475@cornell.edu> 2016-04-22T19:49:40.922Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:56:20.790Z.
% * <akl62@cornell.edu> 2016-03-01T15:44:01.576Z:
%
% > 0.5 log units
%
% I would explain what this is
%Resolved
% ^ <akl62@cornell.edu> 2016-03-15T14:44:55.365Z.

This test may give a rough estimate of coliform concentration, but it can be regarded essentially as a highly accurate presence/absence test as the 100 CFU/mL concentration level is far above what is considered the maximum level for safe drinking water. However, this means that the test is only reliable for relatively high concentrations of bacteria and provides false negatives for concentrations lower than 100 CFU/mL which makes this test impractical for AguaClara use. 
% * <cpf45@cornell.edu> 2016-04-13T21:03:56.489Z:
%
% > highly accurate presence/absence test as the 100 CFU/mL concentration level is far above what is considered the maximum level for safe drinking water.
%
% So it's only 'highly accurate' for high concentrations. But aren't you more interested in detecting ANY concentration? Even a low concentration could negatively impact people drinking the water.
%
% ^ <jmd475@cornell.edu> 2016-04-22T14:49:47.956Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:57:14.265Z.

\subsection*{\textit{Motility of E-coli}}
The team looked into the idea of a sliding colony counter, or a tube in which a water sample and media is placed in. The coliform and E-coli colonies would change color under UV light, which would make these colonies easier to identify. A sliding colony counter, or an electronic device used to quantify colonies, would then slowly slide down the tube, counting colonies. To look into the possibility of this method, information was required on the swimming speed of E-coli. E-coli swims when exposed to chemicals it are attracted to, including amino acids and sugars. This process is called chemotaxis \cite{mittal_motility_2003}. A study done at Harvard University found that an E-coli cell swam at an average speed of 27 µm (micrometers/s) \cite{mittal_motility_2003}. This study grew E-coli in agar and placed the mixtures on glass slides to track the speed of E-coli. This speed was consistent to E-coli swimming 1-2 body lengths per second. The speed was converted to 2.33 m/day. This experiment showed the high speed of E-coli and the difficulty of identifying colonies in a tube due to this rate. Since E-coli swims at a high speed, it would be difficult to isolate individual colonies and count them. For AguaClara purposes, this method of using a sliding colony counter would not be ideal unless the motility of E-coli was reduced.
% * <akl62@cornell.edu> 2016-05-03T15:01:05.909Z:
%
% > difficulty of identifying colonies
%
% I would explain with more detail why faster E-coli means its difficult to count colonies
%
% ^ <akl62@cornell.edu> 2016-05-14T16:55:59.281Z.
% * <akl62@cornell.edu> 2016-05-03T14:59:48.140Z:
%
% >  27µ/s 
%
% is this micrometers per secord or another unit?
%
% ^ <jhs373@cornell.edu> 2016-05-06T19:06:12.084Z:
%
% Yes
%
% ^ <akl62@cornell.edu> 2016-05-14T16:54:56.487Z:
%
% microliters is volume per second which is a flow rate. Either make it say average flow or average speed and change units.
%
% ^ <jhs373@cornell.edu> 2016-05-16T00:30:05.064Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-19T20:04:32.717Z.
% * <akl62@cornell.edu> 2016-05-03T14:58:53.619Z:
%
% > sliding colony counter
%
% Is this a device or what?
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:45:13.882Z:
%
% Yes
%
% ^ <akl62@cornell.edu> 2016-05-19T20:05:37.678Z.
% * <akl62@cornell.edu> 2016-05-03T14:57:49.416Z:
%
% >  is 
%
% are
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:46:04.567Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:56:03.499Z.
% * <cpf45@cornell.edu> 2016-04-13T21:17:06.756Z:
%
% > individual colonies
%
% Individual cells are different from colonies.
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:34:21.976Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:59:18.889Z.
% * <cpf45@cornell.edu> 2016-04-13T21:16:43.086Z:
%
% >  E-coli 
%
% They looked at individual cells?
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:34:25.224Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T14:59:26.005Z.
% * <cpf45@cornell.edu> 2016-04-13T21:13:35.255Z:
%
% > 1.72 m/day
%
% How did you get this number from 27 um/s? I got a bigger number
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:34:29.040Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:00:09.469Z.
% * <cpf45@cornell.edu> 2016-04-13T21:07:49.584Z:
%
% >  sliding bacteria colony counter, 
%
% Describe what this is.
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:44:46.791Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-19T20:05:41.137Z.
\begin{figure}[H]
% * <cpf45@cornell.edu> 2016-04-13T21:18:53.417Z:
%
% Nice figure, but do you ever mention it in the text?
%
% ^ <akl62@cornell.edu> 2016-05-19T20:05:45.317Z.
\centering
\includegraphics[scale=0.4]{motility.jpg}
% * <akl62@cornell.edu> 2016-05-03T15:09:29.310Z:
%
% > s[scale=0.25]{motility.jpg}
%
% Make this bigger
%
% ^.
\caption{E-coli}
\label{Motility}
\end{figure}
% * <akl62@cornell.edu> 2016-03-01T15:45:57.479Z:
%
% >  identifying individual colonies in a tube due to this rate.
%Resolved
% So does that mean the slider approach is not feasible or just more difficult in regards to trying it out in aguaclara?
%
% ^ <akl62@cornell.edu> 2016-03-15T14:46:14.548Z.

\subsection*{\textit{Lactose Fermentation as Indicator of Total Coliforms}}
One of the most commonly used indicator media for the identification of pathogenic E. coli (E. coli O157:H7) is the Sorbitol MacConkey Agar supplemented with Cefixime and potassium-tellurite (SMAC).  This uses sorbitol instead of the lactose used by traditional MacConkey agar to identify bacteria that can ferment sorbitol and those that cannot. Because non-pathogenic E. coli can ferment sorbitol and O157:H7 cannot, the acidic environment created by the fermentation of sorbitol and the basic environment created by the use of peptones as a food source change the color of the included pH indicator neutral red. SMAC agar Cefixime and potassium-tellurite allow for the selective growth of E. coli O157:H7. This prevents false positives from other non-sorbitol fermenting bacteria but also inhibits the growth of other strains of E. coli. (Muller 2005). There is no evidence that the response of pathogenic strains of E. coli to water treatment processes is different from the responses of other strains of E. coli. Therefore the WHO has determined that conventional testing , such as using MacConkey agar,  is sufficient for indicating the pathogen content of drinking water \cite{world_health_organization_guidelines_2011}. A single SMAC plate costs \$17.87 and must be stored at a temperature between 2 and 8 degrees Celsius, and therefore this media would not be suitable for the purposes of this test. The idea behind the traditional MacConkey agar – that lactose fermenting bacterial colonies create an acidic environment and therefore can be identified with a pH indicator – is not completely unusable. Most total coliforms have the ability to ferment lactose at high temperatures around 30-35 C and therefore could be identified using this technique. \cite{world_health_organization_guidelines_2011}
% * <akl62@cornell.edu> 2016-05-03T15:04:30.419Z:
%
% > O15
%
% Is this O or 0. Be consistent
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:50:07.341Z:
%
% O, Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:56:21.397Z.
% * <akl62@cornell.edu> 2016-05-03T15:03:22.894Z:
%
% > O157:H7
%
% What is this?
%
% ^ <akl62@cornell.edu> 2016-05-03T15:03:38.648Z.
% * <cpf45@cornell.edu> 2016-04-14T12:42:26.540Z:
%
% > high temperatures
%
% What is the range of temperatures? I'm assuming around 30-35C?
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:50:22.996Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:06:17.600Z.
% * <cpf45@cornell.edu> 2016-04-14T12:41:31.223Z:
%
% > conventional testing (including tests that do not detect pathogenic strains such as O157:H7)
%
% What do you mean by conventional testing? Is MacConkey agar the convention??
%
% ^ <jhs373@cornell.edu> 2016-04-22T21:06:18.657Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:06:19.333Z.

\subsection*{\textit{Colitag}}
Another method that has been used extensively for water safety testing is Colitag. The way results are obtained through Colitag is similar to the compartmentalized bag test. The components of the test are added to a 100 ml sample, and after an incubation time, the sample changes to a yellow color indicating the presence of coliforms. Then, an ultraviolet light is used to check for fluorescence, which indicates the presence of E-coli. The incubation time for Colitag is reported to be 16-22 hours, which is shorter than other existing tests, and the incubation temperature was reported as 35 degrees C \cite{hach_modified_2016}. This temperature is higher than that of Honduras, but Colitag is simple to use in a low resource setting and has a short incubation time. The test is cheap as well, but can only be bought in bulk. Therefore, Colitag is an inexpensive option that fits the majority of requirements for an optimal test. 

\noindent The figure below shows the results of a Colitag test, where the yellow color indicates the presence coliforms and the fluorescence indicates the presence of E-coli.
% * <cpf45@cornell.edu> 2016-04-14T12:59:18.491Z:
%
% > were
%
% Did you actually do this? If not it should be in present tense.
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:25:01.427Z:
%
% We have not, resolved and in present tense
%
% ^ <akl62@cornell.edu> 2016-05-03T15:07:44.850Z.
% * <cpf45@cornell.edu> 2016-04-14T12:44:40.425Z:
%
% > a
%
% an
%
% ^ <jhs373@cornell.edu> 2016-04-22T20:24:47.755Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:08:12.841Z.
% * <cpf45@cornell.edu> 2016-04-14T12:44:18.306Z:
%
% > after an incubation time
%
% how long, at what temperature?
%
% ^ <cpf45@cornell.edu> 2016-04-14T12:46:07.650Z:
%
% I see you say it later. Put the incubation time and temperature with the rest of the procedure.
%
% ^ <akl62@cornell.edu> 2016-05-03T15:08:37.066Z.
\begin{figure}[H]
% * <cpf45@cornell.edu> 2016-04-14T12:47:06.836Z:
%
% Again, nice figure but you never mention it in the text.
%
% ^ <akl62@cornell.edu> 2016-05-03T15:08:48.425Z.
\centering
\includegraphics[scale=0.25]{colitag.jpg}
\caption{Colitag Test}
\label{Colitag}
\end{figure}
% * <akl62@cornell.edu> 2016-03-15T14:40:29.304Z:
%
% For each of these tests, I would include some sort of picture or diagram for how each of these tests work. That way readers can see all the different types along with reading about them. 
%
% ^ <jhs373@cornell.edu> 2016-04-07T19:54:03.285Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T16:56:31.373Z.
% * <akl62@cornell.edu> 2016-02-24T05:15:15.545Z:
%
% Overall good literature review! Make sure to read over what you write for grammar mistakes and always try to write as concise as possible. You as little amount of words as you can to get your point across. I would recommend just reading over it again to fix any mistakes and such.
%
% ^ <akl62@cornell.edu> 2016-03-15T14:48:26.587Z.
% * <akl62@cornell.edu> 2016-02-24T05:16:39.615Z:
%
% Also make sure that when you fix a suggestions from a comment, that you reply with "Resolved" or something and not close it so I can look over it again.
%
% ^ <akl62@cornell.edu> 2016-03-15T14:48:35.563Z.

\subsection*{\textit{Loop-mediated Isothermal Amplification Method}}
The loop-mediated isothermal amplification, also known as the LAMP method is a method for amplifying bacterial DNA without the controlled fluctuation of temperature. Typical polymerase chain reactions used to replicate DNA require heating and cooling to allow denaturation, elongation and annealing of DNA by enzymes called polymerases and ligases. This kind of temperature control requires a laboratory grade machine and would be too costly for AguaClara plants. However, Kaneko et. al. have found that the LAMP method is just as effective in amplification as PCR. The elimination of the temperature fluctuations would reduce costs by also eliminating the need for a temperature controlling machine. The DNA could just be incubated at one temperature and there would be no side effects to the cloning. This method would also eliminate the danger of killing bacteria through heating and thus denaturing. On top of this benefit, the LAMP method also loops multiple copies of the DNA to reduce the need for primers which are typically required at the starting ends of the DNA to begin the replication process. This LAMP method in conjunction with EvaGreen dye which attaches to DNA and fluoresces could potentially be another form of inexpensive coliform indication since the only requirements seem to be the enzymes and the EvaGreen dye. 

\section*{Methods and Discussion}
\subsection*{\textit{First Iteration}}
\subsubsection*{Initial Research}
% * <akl62@cornell.edu> 2016-05-14T16:58:19.402Z:
%
% > Initial Research
%
% This paragraph should not be about previous research, but almost like a mini abstract of what you did in this iteration. For example, something like "The purpose of this iteration... create a media, come up with ideas for the sliding colony counter etc. And do this for all iterations
%
% ^ <jmd475@cornell.edu> 2016-05-18T17:12:06.004Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T01:56:39.485Z.
The purpose of this initial step was to use the literature research done prior in order to design the goal product and also design experiments to test the viability of said design. This required the preparation of the proper media and other materials.
\subsubsection*{\textit{Research Application Design}}
Upon gaining an understanding of microbial detection in water, the team went into a brainstorming phase of hatching ideas for potentially viable forms of microbe detection. The overarching goal of a tube form incubator and colony counter led the team to further research on E. coli motility and growth. Figure \ref{colony counter} is the rough design of the sliding colony counter. The tube would have thickened Colitag media and the coliform colonies would turn bright green and the E. Coli colonies would turn blue under UV light. The sliding colony counter, which works by counting individual bacterial colonies, would start on one side of the tube and slowly slide down the tube to count how many colonies of each microbe are present throughout the tube.  Research was done on types of fluid media in which cultures could grow without stunted growth or cell death; hence, a media thick enough to prevent cells from swimming too far from the origin point and allows for segregation of colonies but thin enough to allow unrestricted colony growth in the media became the goal. The media used for the Colitag indicator turns bright green in the presence of fecal coliforms and glows blue under ultraviolet light in the presence of E. Coli. A combination of a semi-gel-like matrix and this color changing media remains the goal to allow for visible and distinguishable coliform and E. Coli colonies. 
% * <akl62@cornell.edu> 2016-05-03T15:13:03.938Z:
%
% > The sliding colony counter
%
% Define this above and also on the image. Is this the device on the right side of the tube?
%
% ^ <jhs373@cornell.edu> 2016-05-10T16:33:13.097Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T01:56:43.354Z.
% * <akl62@cornell.edu> 2016-05-03T15:11:31.332Z:
%
% >  Figure 4 i
%
% This is the wrong figure number. Format it so it adjusts when you add new figures
%
% ^ <akl62@cornell.edu> 2016-05-14T16:57:33.028Z.
% * <cpf45@cornell.edu> 2016-04-14T15:25:39.800Z:
%
% >  bright green
%
% You said yellow before...
%
% ^ <jhs373@cornell.edu> 2016-04-22T21:07:05.433Z:
%
% I think this is in reference to under a UV light while the yellow before was in general
%
% ^ <akl62@cornell.edu> 2016-05-14T16:57:36.565Z.
% * <cpf45@cornell.edu> 2016-04-14T15:23:41.211Z:
%
% > tube form incubator and colony counter
%
% Good place to mention your figure. Also a more in-depth description would help the reader understand what you are envisioning.
%
% ^ <jmd475@cornell.edu> 2016-04-22T20:13:45.583Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:15:06.778Z.

\begin{figure}[H]
\centering
\includegraphics[scale=0.50]{ProductDesign.png}
\caption{Sliding Colony Counter Design}
\label{colony counter}
\end{figure}
% * <akl62@cornell.edu> 2016-05-03T15:15:43.248Z:
%
% > \subsection*{Second Iteration
%
% I would give a brief description of this iteration
%
% ^ <jhs373@cornell.edu> 2016-05-09T19:59:46.703Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T17:01:15.173Z.
\subsection*{Second Iteration}
\subsubsection*{\textit{Description}}
The goal of this iteration of our experiment was to test how different quantities of gelatin affected the viscosity of media. 
% * <akl62@cornell.edu> 2016-05-14T17:01:49.907Z:
%
% > specific
%
% remove
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:38:26.462Z:
%
% Resolved
%
% ^.
\subsubsection*{\textit{Materials}}
\begin{itemize}
  \item 4 g of NaCl
  \item 4 g of Tryptone
  \item 2 g of Yeast Extract
  \item 4 Paragon Jars
  \item 1 Hot Plate
  \item Water sample from Beebe Lake
  \item Styrofoam Box Incubator
  \item 8.5 g of gelatin
\end{itemize} 
\subsubsection*{\textit{Viscosity Experimentation Procedure}}
The goal of the sliding colony counter required the development of a viscous media to incubate the water sample with its potential microbes. However, this method had to be tested for its viability to determine whether or not the survival rate of cells would be affected by the heat of the melted gelatin, which was used for thickening the liquid. Dead bacteria would not be able to grow and thus there would be nothing to detect. The gelatinous samples were tested against a sample in liquid Luria Bertrani broth which is a basic media that fosters cell growth. Each 100ml sample of water had 1 g tryptone, 0.5 g yeast extract, and 1 g NaCl added to provide the bacteria with necessary nutrients to grow in the LB broth. The viscous media had the same amounts of chemicals, however there was melted gelatin added and mixed thoroughly to obtain even thickening. Three different amounts of gelatin (1g, 2.5g, 5g) were added to the samples and each of these amounts of gelatin were melted in 5ml, 5ml and 10ml of heated tap water respectively. The gelatin was melted prior to mixing with the contaminated water samples and broth. The water samples from Beebe lake were assumed to be contaminated such that it would contain cells. The samples were then incubated for 16-20 hours in a styrofoam box. 
% * <akl62@cornell.edu> 2016-05-20T02:15:51.948Z:
%
% > The materials used for this iteration were the same as those of the first. The viscous media had the same amounts of chemicals,
%
% Rewrite this with the original sentence you had. The one with the makeup of the borth
%
% ^ <akl62@cornell.edu> 2016-05-20T02:16:44.739Z:
%
% broth
%
% ^.
% * <akl62@cornell.edu> 2016-05-14T19:23:35.046Z:
%
% > Each 100ml sample of water had 1 g tryptone, 0.5 g yeast extract, and 1 g NaC
%
% Make a bulleted list of all the materials used for each iteration. Just mention new materials used for each subsequent iteration, you don't have to repeat materials
%
% ^ <akl62@cornell.edu> 2016-05-14T19:25:02.970Z:
%
% but say "new materials" if you only decide to write down new matierals.
%
% ^ <jmd475@cornell.edu> 2016-05-18T17:04:49.975Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:15:02.293Z.
% * <akl62@cornell.edu> 2016-05-14T17:07:57.468Z:
%
% > a sample in liquid Luria Bertrani broth
%
% Mention this is a control
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:38:09.939Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:15:33.964Z.
% * <akl62@cornell.edu> 2016-05-14T17:02:45.950Z:
%
% > he final
%
% Don't say final, just say a goal
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:38:16.108Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:15:36.868Z.
% * <akl62@cornell.edu> 2016-05-03T15:21:49.851Z:
%
% > The unheated liquid broth sample was used to confirm the presence of cells in the water sample. 
%
% The procedure seems a little unclear. I would reword it. I'm guessing that the gelatin was mixed with the contaminated water and heated, and then the broth was added in after? Or what?
%
% ^ <jhs373@cornell.edu> 2016-05-09T20:02:48.415Z:
%
% For this iteration, the gelatin was melted separately and then added to the broth and contaminated water
%
% ^ <akl62@cornell.edu> 2016-05-14T17:06:59.920Z.
% * <akl62@cornell.edu> 2016-05-03T15:21:06.039Z:
%
% >  in a styrofoam box. 
%
% Picture with samples in it?
%
% ^ <akl62@cornell.edu> 2016-05-14T17:07:29.449Z.
% * <akl62@cornell.edu> 2016-05-03T15:20:32.685Z:
%
% > heated wate
%
% heated contaminated water or tap water?
%
% ^ <jhs373@cornell.edu> 2016-05-08T22:02:53.297Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T17:07:10.349Z.
% * <akl62@cornell.edu> 2016-05-03T15:19:48.890Z:
%
% >  5ml, 5ml and 10ml 
%
% did you mean to say 5 mL twice?
%
% ^ <jhs373@cornell.edu> 2016-05-06T19:14:19.584Z:
%
% Yes
%
% ^ <akl62@cornell.edu> 2016-05-14T17:07:12.130Z.
% * <akl62@cornell.edu> 2016-05-03T15:16:53.503Z:
%
% >  final goal 
%
% Final goal? 
%
% ^ <jhs373@cornell.edu> 2016-05-09T20:14:24.681Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T17:02:35.215Z.
\subsubsection*{Results}
As expected, the control grew colonies, verifying that the water sample had bacteria. The colonies were mostly floating on the surface and along the walls. The 1 g gelatin sample had a thick layer of gelatin on the bottom and it seemed that the gelatin was holding the colonies at the bottom. The 2.5 g gelatin sample had a ball of gelatin at the bottom of the bottle. The ball of gelatin had many air bubbles and the turbidity of the sample around the gelatin seemed to indicate the presence of many but indistinguishable colonies. The 5 g gelatin sample had a layer 1.25 cm thick of gelatin on the bottom with many air bubbles. The entire surface seemed to have colonies growing. This indicated that there was too much gelatin in the mixture, or that the gelatin was not mixed well with the water sample and media.  The gelatin was found in clumps because it was at a high temperature when mixed with the water samples and media, which were at a low temperature. This experiment was repeated to attempt to replicate or better our results. For the next iteration, the gelatin was mixed more uniformly with the media to create a thickened media.
% * <akl62@cornell.edu> 2016-05-14T17:10:01.822Z:
%
% > This experiment must be run again to attempt to replicate or better our results. For the next iteration, the gelatin will be
%
% keep in past tense
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:39:29.606Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:16:51.489Z.
% * <akl62@cornell.edu> 2016-05-14T17:09:34.005Z:
%
% > experiment must be run again
%
% Repeated.
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:39:33.161Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:17:05.073Z.
% * <akl62@cornell.edu> 2016-05-14T17:08:22.140Z:
%
% >  1g
%
% Add spaces between the number and g for all units
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:39:55.249Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:17:11.239Z.
% * <akl62@cornell.edu> 2016-05-03T15:24:39.605Z:
%
% >  replicate or better our results
%
% Good method, but add more pictures of the bacteria colonies and media and such. And make a table for each trial that gives a brief description of each of the gelatin samples.
%
% ^ <jmd475@cornell.edu> 2016-05-18T17:14:02.459Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:17:20.029Z.

Another issue that arose was that an extra 5 ml of water was added to the 5 g gelatin sample to ensure that the gelatin completely dissolved. Thus, the volume of each sample was not the same. This resulted in a lack of continuity between samples in the experiment, and may have resulted in a  different result for the 5 g gelatin mixture. The experiment will be attempted again to solely test the amount of gelatin in the mixture rather than the volume of the mixture.
% * <akl62@cornell.edu> 2016-05-14T19:01:22.736Z:
%
% > This resulted in a lurking variable that may have led to a different result with the 5 g gelatin mixture. The volume was supposed to remain constant for each mixture, but ended up varying.
%
% Reword this. Remove lurking variable. Say something like lack of continuity between samples in the experiment
%
% ^ <jhs373@cornell.edu> 2016-05-16T01:15:45.606Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:17:33.120Z.

\begin{table}[H]

\begin{center}
\caption{Second Iteration}
  \label{tab:table1}
  \begin{tabular}{| l | c |}
  	\hline
    Jar & Observations/Results\\
    \hline
    Control &   Microbes grew on sides of bottle.\\
    \hline
    1g gelatin &  Microbes on the sides of bottle as well the bottom\\
    \hline
    2.5g gelatin & Semi-thick layer of gelatin and microbes at bottom or jar\\
    \hline
    5.0g gelatin & 1.27 cm thick layer of gelatin and microbes on bottom of jar\\
    \hline
  \end{tabular}
\end{center}
\end{table}
 
 
\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2510.JPG}
\caption{All viscosity experiment samples after incubation}
\label{2group}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2512.JPG}
\caption{Bottom layer of sample with 5 g of gelatin}
\label{2-5}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2513.JPG}
\caption{Bottom layer of sample with 2.5 g of gelatin}
\label{2-2.5}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2514.JPG}
\caption{Bottom layer of sample with 1 g of gelatin}
\label{2-1}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2515.JPG}
\caption{Bottom layer of control sample}
\end{figure}

\subsection*{Third Iteration}
\subsubsection*{\textit{Description}}
For this iteration, the media containing the water samples was heated up before the gelatin was added. The goal of this technique was to ensure a more uniform mixture of gelatin and media. Results were also taken after both 24 and 48 hours to determine an ideal incubation time.
% * <akl62@cornell.edu> 2016-05-14T19:04:01.013Z:
%
% > This iteration focused on a different technique for the same experiment.
%
% Remove or make more concise
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:40:26.978Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:18:42.173Z.
\subsubsection*{\textit{Procedure}}
This procedure followed almost the exact procedure as in the second iteration. No new materials were required for this iteration. The contents were mixed once more in four paragon jars. One jar was left with only the broth as a control, and placed in the styrofoam box incubator. The control jar was heated up in future iterations. In the other three jars, the thickened media was set up. However, for this iteration, the media was heated up to 38 C and then mixed with 1 g, 2.5 g, and 5 g of gelatin, respectively. This allowed the gelatin to melt while mixing with the media. The maximum tolerable temperature for E. Coli was found to be 45 C thus the temperature of the media was measured constantly to ensure that this critical temperature was never exceeded during the heating process. Then after mixing in the gelatin, the samples were heated up again to let the gelatin dissolve better and which removed some gelatin from the walls to prevent bacterial growth on the walls as was the case in the previous iteration. The goal of this was to ensure a more uniform mixture of gelatin and broth. 
% * <akl62@cornell.edu> 2016-05-14T19:05:43.220Z:
%
% >  One jar was left with the broth as a control,
%
% Were all four jars kept at the same temperature during incubation, because you said you only heated up the jars with the media. They should all be the same temperature during incubation
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:44:49.943Z:
%
% For this iteration, we actually erraneously did not heat up the control. We fixed this in the next iteration.
%
% ^ <akl62@cornell.edu> 2016-05-20T02:22:08.766Z.
\subsubsection*{\textit{Results}}
The results of this iteration was very informative in that it indicated that the 5 g gelatin after only 24 hours worked the best in achieving the goal of isolating colonies as seen in Figure \ref{3-5}. Although some of the colonies combined together to form large groups of CFUs, there were visible chunks of bacteria at the bottom of the bottle. However, most of the liquid was in a sense wasted since it was not thickened in any way. Only a thin (1.27 cm) layer resting on the bottom of the bottle was thickened and the rest of the liquid media was simply sitting on top. The same observations were made for the other samples as well with thinner bottom gelatinous layers. The 1 g bottle at fist glance looked nearly identical to the control, but the bottom of the bottle seemed to have more bacteria observed through general turbidity. However, there were no defined colonies. Another difference between the 1 g and the control was that when the 1g bottle was tipped over a few degrees, the bottom layer seemed to stay together as opposed to the control where the bacteria would just scatter through the liquid. the 2.5 g had a bottom layer similar to the 1 g except that the layer itself was slightly thicker. Figure \ref{3group} is a side by side comparison of the samples after 24 hours of incubation wherein the layers of gelatin are visible on the bottom of the bottles as described earlier.
% * <akl62@cornell.edu> 2016-05-14T19:18:06.269Z:
%
% > ayers of gelatin are visible on the bottom of the bottles
%
% Having trouble seeing the layers. Maybe put an arrow on the picture to show where they are 
%
% ^ <jmd475@cornell.edu> 2016-05-18T17:39:28.051Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:24:28.094Z.
\newline After 48 hours of incubation, the 5 g bottle no longer had distinguishable colonies for the proliferation of the bacteria grew to be too much to the point of complete amalgamation of all colonies. The control bottle had more of an overall turbid look after 48 hours rather than a thin layer on the bottom. On the other hand, the 1 g bottle still maintained its layer of bacteria and gelatin on the bottom. The layer did not grow any thicker but rather more turbid. 
\newline Possibly due to the difference in the method for mixing gelatin, there were almost no bacteria growing on the walls of the control nor the 1 g gelatin bottles. The previous method did not have a second heating and swirling cycle, and did not allow the gelatin stuck on the walls to melt off thus facilitated bacterial growth on the walls.  
% * <akl62@cornell.edu> 2016-05-14T19:11:07.755Z:
%
% > Possibly due to the difference in the method for mixing gelatin, there were almost no bacteria growing on the walls of the control nor the 1g gelatin bottles. 
%
% Doesn't bacteria on the walls mean that the bacteria were moving fast?
%
% ^ <jmd475@cornell.edu> 2016-05-18T17:42:16.899Z:
%
% we think that because we swirled the bottles after heating then reheated and swirled again to let the gelatin melt off the walls, there was no growth on the walls this time. The first time we did it there were still some gelatin granules left on the walls which probably facilitated the growth (you can kind of see the granules in the pictures). Decided to put this explanation in the paper itself, thanks
%
% ^.


\begin{table}[H]

\begin{center}
\caption{Third Iteration}
  \label{tab:table2}
  \begin{tabular}{| l | c | c |}
  	\hline
    Jar & 24 hours & 48 hours\\
    \hline
    Control &   Microbes throughout bottle & No change\\
    \hline
    1g gelatin &  Thin bottom layer of microbes & Maintained layer\\
    \hline
    2.5g gelatin & Semi-thick bottom layer & Maintained layer\\
    \hline
    5.0g gelatin & Thick bottom layer & Layer lost thickness\\
    \hline
  \end{tabular}
\end{center}
\end{table}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_9219.jpg}
\caption{Profile view of all bottles after 24 hours of incubation. Note the gelatin layers on the bottom of the jars for 1g, 2.5, and 5g gelatin.}
\label{3group}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.250]{5g_gelatin.JPG}
% * <akl62@cornell.edu> 2016-05-14T19:08:45.878Z:
%
% > hics[scale=0.10]{5g_gelatin.JPG}
%
% Make this image bigger so we can see it!
%
% ^.
\caption{Bottom view of the 5g jar after 24 hours incubation. There are visible flocs of colonies dispersed all around the bottom.The white dots are bacterial growths.}
\label{3-5}
\end{figure}

\subsection*{Fourth Iteration}
\subsubsection*{\textit{Description}}
This iteration focused on testing smaller quantities to observe whether results remained consistent. For this iteration, petri dishes were used to mix media and gelatin. The goal of this experiment was to create a thick media throughout the petri dish. On top of that, the control jar was heated along side the other samples in order to maintain consistency throughout all samples. The previous iteration did not take into account that the heating of the samples could kill off some bacteria, thus the unheated control bottle would not have been an accurate representation of the number of cells that would have grown without gelatin. Measures were taken to ensure the uniformity of the experiment.
\subsubsection*{\textit{New Materials}}
\begin{itemize}
\item 2 Petri Dishes
\end{itemize}
\subsubsection*{\textit{Procedure}}
The protocol for the 95 ml samples was nearly identical to that of the third iteration. The only difference was that the control 0 g gelatin jar was also heated as a precautionary measure for consistency across the board. 
As for the two petri dishes, 1 g of gelatin was added to a total of 16 ml of water. 15 ml of water was used for dissolving the gelatin and 1 ml of sample was used for the pour plate method. The pour plate method allows for the plating of liquid samples with colonies growing within the gelatinous media. Since the 15 ml of tap water was the only liquid heated, the 1 ml sample containing E. Coli was not heated, thus lowered the chances of microbial death. Once the 1 g of gelatin was melted in the 15 ml of water, the 1 ml sample was poured onto the plate, followed by the melted gelatin. Following this swift step, the lid of the petri dish was placed back on the dish and was gently swirled clockwise and counterclockwise before the gelatin hardened. The petri dishes were sealed with tape around the perimeter to ensure minimal contamination. Both the petri dishes and jars were incubated for 24 hours and 48 hours. Although the pour plate method would not have provided the thickness desired, it would have allowed for the isolation of visible colonies, as desired. 
% * <akl62@cornell.edu> 2016-05-14T19:21:56.965Z:
%
% > was added to a total of 16m
%
% mention that the same amount is placed in  both petri dishes
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:55:08.990Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:28:36.546Z.
% * <akl62@cornell.edu> 2016-05-14T19:16:43.649Z:
%
% > allowed provided
%
% provided
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:47:14.655Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:28:39.258Z.
% * <akl62@cornell.edu> 2016-05-14T19:15:36.205Z:
%
% > regular
%
% tap or DI
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:47:48.524Z:
%
% tap
%
% ^ <akl62@cornell.edu> 2016-05-20T02:32:35.796Z.
% * <akl62@cornell.edu> 2016-05-14T19:12:59.405Z:
%
% > pour plate method
%
% Explain briefly
%
% ^.

\subsubsection*{\textit{Results}}
The results of this iteration was very similar to that of the third iteration in the way that the gelatinous layers formed on the bottom of the jars. The results for the 5 g bottle were consistent with those in the third iteration as one could observe by comparing Figure \ref{3group} and Figure \ref{4group}. The bottles in Figure \ref{4group} are much more turbid overall although the gelatin layers are still visible. The petri dishes did not solidify in the way that was expected. Since the gelatin to media ratio was greater than that of the jars, the petri dishes were expected to solidify completely. The contents of the petri dishes were still very fluid and there did not even seem to be a gelatin layer on the bottom as with the bottles. Instead, there were large films of bacterial growth that developed in the media as observable in Figure \ref{4petri}.
% * <akl62@cornell.edu> 2016-05-14T19:19:05.207Z:
%
% > To the team's demise,
%
% Remove
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:48:09.926Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:40:54.157Z.
% * <akl62@cornell.edu> 2016-05-14T19:17:05.653Z:
%
% > as 
%
% with
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:49:32.616Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:40:56.107Z.
\newline In an attempt to obtain only the bottom gelatin layer of the 5 g bottle (figure \ref{4group}), the team poured out the top media, but was unsuccessful since even the gelatin layer could not handle being tipped over above the horizontal position and the gelatin layer was flushed out along with the rest of the media. From these results, the team attempted another iteration to test only the thickening of water without media or sample water with bacteria. 

\begin{table}[H]

\begin{center}
\caption{Fourth Iteration}
  \label{tab:table3}
  \begin{tabular}{| l | c | c |}
  	\hline
    Jar & 24 hours & 48 hours\\
    \hline
    Control &  Microbes on walls & No change\\
    \hline
    1g gelatin &  Thin bottom layer of microbes & Maintained layer\\
    \hline
    2.5g gelatin & Semi-thick bottom layer & Layer was more motile\\
    \hline
    5.0g gelatin & Thick layer with visible colonies& Layer lost thickness\\
    \hline
    Petri Dish 1 & Visible colonies in thick layer & Less thick  \\
    \hline
    Petri Dish 2 & Heavy condensation on lid & no solidification \\
    \hline
  \end{tabular}
\end{center}
\end{table}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_2580.JPG}
\caption{Bottom view of the 5g bottle after 24 hours of incubation. Again, there are visible colonies formed on the bottom of the jar, much like those of the 5g jar of the third iteration.}
\label{4-5}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{4group.JPG}
\caption{Profile view of all bottles. From left to right: Control, 1g gelatin, 2.5g gelatin, 5g gelatin. Note the layers on the bottom of the jars for 1g, 2.5, and 5g gelatin and the overall turbidity of the control jar.}
\label{4group}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.25]{5g_gelatin__1_.JPG}
\caption{Bottom view of the 5g gelatin bottle after 48 hours of incubation. The bottom of the jar is more turbid overall and the individual colonies that were able to be seen before are no longer distinguishable.}
\label{4-5-48}
\end{figure}


\begin{figure}[H]
\centering
\includegraphics[scale=0.10]{IMG_9264.JPG}
\caption{Top view of the petri dishes. There is a lot of condensation visible on dish 2 and the white bacterial films are visible in dish 1.}
\label{4petri}
\end{figure}


\subsection*{Fifth Iteration}
\subsubsection*{\textit{Description}}
After experimenting with growing bacteria in media with different concentrations of gelatin, the team took a step back to focus on just gelatin. While the gelatin thickened media at the bottom of the jars in the previous iterations, the mixture was not uniformly thickened. This iteration focused on determining the necessary gelatin concentration to completely thicken a liquid.
\subsubsection*{\textit{Materials}}
\begin{itemize}
\item ProCoDA thermometer
\item Tap Water
\item Paragon Jars
\item Styrofoam box

\end{itemize}
\subsubsection*{\textit{Experimenting with only Gelatin Procedure}}
This procedure required just tap water, gelatin, and the paragon jars used in the previous procedures. To test how effective different amounts of gelatin were for liquid, smaller quantities of liquid were used. 20 ml of water were added to each of four paragon jars. 20 ml were used as opposed to 100 ml to use smaller quantities of gelatin. The ratio of gelatin to water would be the same for 100 ml of water, however. For example, 1 g of gelatin in 20 ml of water would be equivalent to 5 g of gelatin in 100 ml of water. The amounts of gelatin used in each bottle were 1 g, 2.5 g, 3.75 g, and 5 g, respectively. The jars were heated to 38.5C, while ProCoDA was used to track the temperature over time. At this temperature, the gelatin was mixed with the water and allowed to melt. Once the gelatin had melted, the jars were placed in the styrofoam box incubator to cool for 24 hours. 
% * <akl62@cornell.edu> 2016-05-14T19:26:07.056Z:
%
% >  its
%
% the
%
% ^ <jhs373@cornell.edu> 2016-05-14T23:51:11.026Z:
%
% Resolved
%
% ^.
% * <akl62@cornell.edu> 2016-05-14T19:25:51.641Z:
%
% >  the
%
% remove
%
% ^ <jhs373@cornell.edu> 2016-05-16T01:16:52.734Z:
%
% Which one?
%
% ^ <jhs373@cornell.edu> 2016-05-16T01:17:52.556Z:
%
% Never mind, resolved
%
% ^.

\subsubsection*{\textit{Results}}
The results of the fifth iteration showed the viability of gelatin as a thickener. All four jars had a solid mixture. Therefore, all four quantities of gelatin worked well at thickening liquid. One result was surprising, however, as the 1 g of gelatin thickened 20 ml of water, but 5 g of gelatin could not completely thicken 100 ml of media in the previous iterations. This leads to a question on whether gelatin thickens water and media differently. This question leads to the next iteration.

\begin{table}[H]

\begin{center}
\caption{Fifth Iteration}
  \label{tab:table4}
  \begin{tabular}{| l | c |}
  	\hline
    Jar & 24 hour Results \\
    \hline
    1g gelatin &  Thick layer of gelatin \\
    \hline
    2.5g gelatin &  Thick layer of gelatin \\
    \hline
    3.75g gelatin & Thick layer of gelatin \\
    \hline
    5.0g gelatin & Layer was very thick: difficult to remove from jar\\
    \hline
  \end{tabular}
\end{center}
\end{table}

\begin{figure}[H]
                                                      \centering
\includegraphics[scale=0.20]{5group.jpg}
\caption{Side-by-side comparison of the bottles. Increasing gelatin concentration increases the darkness of the gel while thickening the sample more as well.}
\label{5group}
\end{figure}


\subsection*{Sixth Iteration}
\subsubsection*{\textit{Description}}
This purpose of the sixth iteration was to test whether gelatin thickens water and media differently. In the previous iteration, 1 g of gelatin was enough to thicken 20 ml of water. This iteration replicated the previous procedure, but with 20 ml of LB broth.

\subsubsection*{\textit{New Materials}}
\begin{itemize}
\item Petri Dish
\item Three Paragon Jars
\item Ingredients for LB Broth
\end{itemize}

\subsubsection*{\textit{Experimenting with Gelatin in Media Procedure}}
This procedure was very similar to the previous procedure. For this iteration, only two quantities of gelatin were tested. From the previous iteration, it was determined that 3.75 g and 5 g of gelatin were higher quantities of gelatin that necessary for thickening 20 ml of liquid. Therefore, only 1 g and 2.5 g were tested. A petri dish was also included in this experiment to observe the thickened media in a plate. The same procedure as the second, third, and fourth iterations was used to create the LB Broth. 20 ml of broth and tap water were then added to three jars. One of these jars was used for the petri dish, as a petri dish cannot be heated using a hot plate. The three jars were heated to approximately 38.5 C, and then 1 g, 2.5 g and 1 g of gelatin were added to each, respectively. The temperature was once again measured using ProCoDA. After the gelatin melted in the broth, the contents of the third jar were poured into the petri dish. The two other jars and petri dish were then placed in the styrofoam box to cool for 24 hours. 
% * <akl62@cornell.edu> 2016-05-14T19:28:31.254Z:
%
% > the ProCoDa
%
% This is just "ProCoDA
%
% ^ <jhs373@cornell.edu> 2016-05-16T01:17:33.938Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:48:16.561Z.


\subsubsection*{\textit{Results}}

The results of this iteration were highly informative. Both jars and the petri dish had solid media, which indicated that the gelatin worked regardless of whether being mixed with water or media. Both 1 g and 2.5 g of gelatin thickened 20 ml of media. The 1 g of gelatin also thickened the media in the petri dish. One result that was not expected was the growth of bacterial colonies. As shown in Figure \ref{6-1}, there are many white dots on the bottom of the jar. These dots represent colony forming units, (CFUs), and where easy to distinguish from one another. Therefore, in 20 ml of media that used tap water instead of contaminated water, 1 g of gelatin was able to isolate bacterial colonies. The 2.5 g of gelatin also thickened the media, and created, and as shown in Figure \ref{6-25}, a darker color than the 1 g of gelatin. Only a few bacterial colonies grew in this thickened media. A few bacterial colonies grew in the petri dish. However, these colonies were unexpected since the water used for this experiment were thought to have been sterile. The colonies could very well have been due to contamination of the water or the equipment used. Another iteration could be done in the future to determine the true cause.
% * <akl62@cornell.edu> 2016-05-14T19:33:15.595Z:
%
% > However, these colonies were unexpected since the water used for this experiment were thought to have been sterile. The colonies could very well have been due to contamination of the water or the equipment used.
%
% This is very interesting. Do you think the tap water had bacteria in it? Or do you think it was due to insufficient sterilization of the equipment?
%
% ^.

\begin{table}[H]

\begin{center}
\caption{Sixth Iteration}
  \label{tab:table5}
  \begin{tabular}{| l | c |}
  	\hline
    Jar & 16 hour Results \\
    \hline
    1g gelatin &  Solid layer with lots of CFUs \\
    \hline
    2.5g gelatin & Solid layer with air bubble cluster \\
    \hline
    Petri Dish & Solid, distinguishable CFUs \\
    \hline
  \end{tabular}
\end{center}
\end{table}

\begin{figure}[H]
\centering
\includegraphics[scale=0.14]{6-1.jpg}
\caption{Bottom view of the 1g gelatin bottle with media.The many white dots are colonies that grew throughout the media.}
\label{6-1}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.15]{6-2_5.jpg}
\caption{Bottom view of the 2.5g gelatin bottle with media.No colonies observed. The disturbance in the middle area is a cluster of air bubbles.}
\label{6-25}
\end{figure}

\begin{figure}[H]
\centering
\includegraphics[scale=0.15]{6petri.jpg}
\caption{Top view of the 1g gelatin media petri dish. The visible white dots are bacterial colonies.}
\label{6petri}
\end{figure}
% * <cpf45@cornell.edu> 2016-04-14T15:31:32.698Z:
%
% Have you actually done this? If so, great job!
%
% ^ <akl62@cornell.edu> 2016-05-20T02:48:28.004Z.
% * <cpf45@cornell.edu> 2016-04-14T15:30:36.861Z:
%
% > ~
%
% Proofread your report, this symbol doesn't appear in the text!
%
% ^ <jmd475@cornell.edu> 2016-04-22T20:18:16.984Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-20T02:48:30.045Z.
% * <cpf45@cornell.edu> 2016-04-14T15:29:00.973Z:
%
% > Each 100ml sample of water had 1g tryptone, .5g yeast extract, and 1g NaCl
%
% I didn't realize you were just using these chemicals to make LB broth. You can buy LB broth that you just add water to, in fact there is some in the lab...
%
% ^ <akl62@cornell.edu> 2016-05-20T02:48:34.215Z.


% * <akl62@cornell.edu> 2016-03-15T14:49:27.168Z:
%
% Sounds like you guys have a lot of good research to go off of for this. I recommend you start to design this system, and make a drawing of it, research chemicals and parameters that can be used and such.
%
% Sounds like you guys have a lot of good research to go off of for this. I recommend you start to design this system, and make a drawing of it, research chemicals and parameters that can be used and such.
%
% ^ <jhs373@cornell.edu> 2016-04-07T19:53:55.003Z:
%
% Resolved
%
% ^ <akl62@cornell.edu> 2016-05-03T15:16:38.496Z.

\section*{Conclusion}
% * <akl62@cornell.edu> 2016-05-14T19:53:42.016Z:
%
% > Conclusion
%
% Great Conclusion Guys!
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:15:03.373Z:
%
% woo!
%
% ^.
While keeping the ultimate goal of this team in mind, a few key points in carrying out the bacterial incubation and growth stood out. The optimal incubation time for visible and distinguishable CFUs was 24 hours. 48 hours showed to be too long and allowed multiple CFUs to combine together into larger conglomerates which defeats the team's purpose of quantifying the colonies. The final iteration showed that spreading the media out on greater area such as a petri dish as opposed to allowing thick volumes over a small area as with the jars allowed for less colonial overlap thus CFUs were able to be distinguished from other CFUs. With testing only the gelatin, the team found that 1g of gelatin per 20ml of water was enough and possibly even more than the thickness desired. Any greater gelatin concentration would disrupt the visibility of the CFUs because the color of the media itself became darker. On top of the visibility disadvantages of excess gelatin, the media became more solid and some of the greater gelatin concentrations appeared to be too thick to allow bacterial growth. Iteration six allowed the liquid to thicken even with media. Considering the unexpected bacterial growth in the sixth iteration, petri dishes continued to prove themselves as the better option for a container because petri dishes are sterilized as opposed to the glass jars which are washed by the team members. The contamination of the jars could possibly be avoided by the implementation of an autoclave step in the cleaning process. Autoclaving involves highly pressurized saturated steam to sterilize laboratory equipment. The team was not able to test any color indicators this semester but found a few potential indicators for future semesters.Overall it seems that the team made significant progress in laying out the foundation for future the future of this subteam such that more specific and refined experiments could be carried out in the future.
% * <akl62@cornell.edu> 2016-05-14T19:53:20.241Z:
%
% >  autoclave step
%
% describe briefly
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:28:31.554Z:
%
% resolved
%
% ^.
% * <akl62@cornell.edu> 2016-05-14T19:52:55.985Z:
%
% > media increases and 
%
% Edit
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:30:53.980Z:
%
% resolved
%
% ^.
% * <akl62@cornell.edu> 2016-05-14T19:52:06.073Z:
%
% > Iteration six allowed bacterial growth in both 1g gelatin samples but none in the 2.5g gelatin sample which could possibly have been an indication that 2.5g gelatin was too much for the bacteria to grow.
%
% I wouldn't make this claim since you didn't use contaminated water to test it. It could have been due to contamination of other things
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:28:41.455Z:
%
% resolved
%
% ^.


\section*{Future Work}
% * <akl62@cornell.edu> 2016-05-14T19:55:19.156Z:
%
% > Future Work
%
% Make the goals more defined. It seems like you do a lot of explaining but make sure you discretely say what they are!
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:23:24.501Z:
%
% resolved
%
% ^.
For the future, the primary goal of this subteam will be to move towards the development of the sliding colony counter itself. This semester, the subteam was able to thicken media completely with gelatin. This was one of the goals for this semester, and will lead into next semester's work. The implementation of the colony counting device will be the goal for next semester. Now that gelatin's ability to thicken media as well as the necessary amount of gelatin are known, the next goal would be to use this thickened media to actually count colonies. This would be done using an indicator to make the colonies more visible. One of the first steps for next semester would be to find the proper indicator. Once a suitable indicator is found, the team can move towards the goal of an actual sliding colony counter. If not, different methods will be researched as this subteam is important to ensuring that AguaClara is safe after plant treatment. The top options as of now are the EvaGreen dye and Colitag since they may possibly be affordable. Communication with the sellers will be sought to find out the pricing details. The contamination discovered in the last iteration must be resolved before any experimentation in the future, otherwise future experiments cannot be deemed accurate nor valid. The bacteria is thought to have been from either the tap water itself or residual bacteria left from previous iterations due to insufficient cleaning. Investigating this will most likely be a priority for next semester. 
% * <akl62@cornell.edu> 2016-05-14T19:54:42.328Z:
%
% > AguaClara water is 100 percent safe.
%
% I wouldn't include this in there. there is still some room for error in everything
%
% ^ <jmd475@cornell.edu> 2016-05-18T18:15:47.352Z:
%
% resolved
%
% ^.
% * <cpf45@cornell.edu> 2016-04-14T15:33:46.742Z:
%
% > This research resulted in the possibility of agar or gelatin being used as a thickener, and testing this is the next step for the team. 
%
% So you haven't done the viscosity experiment? If you haven't, that section should not be written in past tense.
%
% ^ <jhs373@cornell.edu> 2016-04-22T21:10:11.044Z:
%
% resolved
%
% ^ <akl62@cornell.edu> 2016-05-14T19:54:38.211Z.

\clearpage


\bibliographystyle{apalike}
\bibliography{Zotero1}

% * <akl62@cornell.edu> 2016-02-24T05:19:20.480Z:
%
% Make sure you have proper formatting for these references. Usually, only the second lines on are indented and the first one is the only one not indented. You can either do it manually in the source code or use the method Will showed in class. 
% Resolved
% ^ <akl62@cornell.edu> 2016-03-15T15:02:22.977Z.

\clearpage

\part*{Semester Schedule}

\subsection*{Task Map}

\begin{figure}[H]
\centering
\includegraphics[scale=0.5]{Task_flow.png}
\caption{Task Map}
\label{Task Map}
\end{figure}

\subsection*{Task List}
 
\begin{enumerate}
% * <akl62@cornell.edu> 2016-03-01T15:50:47.807Z:
%
% > Determine what the purpose of this project is as well as the limitations of the tests that can be run. 
% > \newline Target Date: 2/22/16 by Emily -Completed
%
% Delete this. Just seems like you forgot to because its the same thing as number 4
% Resolved
% ^ <akl62@cornell.edu> 2016-03-15T15:04:57.066Z.
\item Research methods for detecting pathogens using indicator species in drinking water that have been established and accepted by the EPA and WHO. Use resources from the EPA website, the WHO’s “Guidelines for Safe Drinking Water” 4th ed., published papers on microbiological water testing.
\newline Target Date: 2/18/16 by Janak -Completed.
\item  Determine what resources in Honduras are available to use for testing. The tests that can be done experimentally in a lab must be able to be replicated there. 
\newline Target Date:2/18/16 Jacqueline -Completed.
\item Determine what the purpose of this project is as well as the limitations of the tests that can be run. 
\newline Target Date: 2/22/16 by Emily -Completed.
\item Look into the possibility of using a liquid culture colony.The possibility of this method is something to discuss with Monroe.
\newline Target Date: 2/26/16 by Janak -Completed.
\item Compile a list of viable testing methods to research further. This list will be made through comparing indicators, prices, and incubation times of the different tests.  
\newline Target Date: 3/1/2016 by Janak - Completed.
\item Once the list of testing methods has been analyzed completely, begin testing different methods to determine which are the most effective and efficient for the purposes of this project.
\newline Target Date: 3/17/2016 by Jacqueline - Completed.
\item Determine which techniques would be the most effective in meeting the requirements for our test: cheap (less than five dollars per test), functional under a wide range of environmental conditions, and easy to operate and interpret.
\newline Target Date: 4/7/2016 by Jacqueline - Completed.
\item Begin designing our own test based by modifying existing tests, or if the liquid culture colony approach showed promise, develop a new type of test based on this technique. 
\newline Target Date: End of semester by Janak- Completed.
\item Use water with known levels of contamination to test this new method.
\newline Target Date: End of semester by Jacqueline
\end{enumerate}

\textbf{Report Proofreader: Jacqueline Dokko}

\end{document}