Coliform Detection in Food and Water

Bonus Experiment
Jo Hanzelle Tadlas
October 20, 2014
Introduction
Water and Food are needed by humans as much as they need air to breathe. So, food and water needs
to be tested for one to know if pathogenic bacteria are present in a given sample through detecting the
presence of coliforms (indicator of the presence of pathogenic bacteria), reducing or fully eliminating
the chances of causing food borne illnesses to those who consume it.
This bonus experiment aims to help students count microbial colonies properly, and determine the Most
Probable Number (MPN) of microbiological contaminants in a given sample the right way.
Experimental Data
Table 1. Presumptive and Confirmatory test results using the MPN method.
Test Number of positive tubes* MPN/mL
10
-3
10
-4
10
-5
10
-6
10
-7
12, 000
MPN/mL Presumptive 3 1 2 2 1
Confirmatory 3 1 2 2 1
*The 10
0
, 10
-1
, 10
-2
dilutions showed no positive test tubes.
10
-3
plated dilution SPREADER
Discussion
The sample used was a sagot gulaman drink providing no positive tubes on 10
0
, 10
-1
, 10
-2
dilutions, and
3, 1, 2, 2, 1 positive ones for the 10
-3
, 10
-4
, 10
-5
,10
-6
, 10
-7
dilutions respectively on both the presumptive
and confirmatory tests. By observing the rule that one must choose the least dilute tube with the most
number of positive tubes, or having all positive tubes, the first part of the combination for the MPN was
the 10
-3
dilution, and then the next two higher dilutions which provided a 3-1-2 combination. This
combination, when basing on the MPN table, showed that it contained about 120 MPN/ml, multiplied
by dilutions it took to get all positives or to 0.1 (100), the sample would have 12,000 MPN/ml.
Since MPN is an estimation of how many viable cells there are per ml of a given sample, this sagot
gulaman has approximately 12,000 bacterial cells per ml. According to the Food and Drug Agency (FDA)
of the Department of Health, the acceptable level of microorganisms in non-alcoholic beverages (ready
to drink, soft drinks, energy drinks, iced tea), is 10-12 mg/100 ml, and is also based on the Minimum
Requirement for Analysis of Finished Product of Annex J, if Good Manufacturing Practice is followed.
This is 0.010 up to 0.012 grams per 100 ml of sample of a non-alcoholic beverage, which in this case can
be the sagot gulaman. The maximum level by which products are rejected for consumption, on the
other hand, is up to 100 cfu/ml (SPC/APC method).
The given sample had 12,000 MPN/ml, or 120 MPN per 100 ml, this is to coincide with the basis of 100
ml done by the FDA. Given that it had 120 MPN/100 ml, the sagot gulaman would be considered unsafe
for consumption if tested positive for specific pathogenic bacteria as the safe maximum level for non-
alcoholic beverages is 100 cfu/ml.
Microorganisms have intrinsic and extrinsic properties that affect their growth (Jay, 2000). Intrinsic
properties include pH, Moisture content, Oxidation-reduction potential (Eh), Nutrient content,
Antimicrobial constituents and its Biological structures. Extrinsic properties include temperature of
storage, relative humidity of environment, presence and concentration of gases, presence and activities
of other microorganisms.

Sagot gulaman is made up of water, gulaman (gelatin), sugar, dried tapioca pearls (sago), and ice if it is
not cold. Any of these ingredients could have contributed to the growth of microorganisms in the
sample, and the way the drink was prepared. The water could have been contaminated by coliforms
before it was put into the juice, the gulaman, sugar, and sago could also have been exposed to the air
for a long duration, the utensils may have not been washed before it was used on the ingredients, the
surface or table where the gulaman was sliced might have not been cleaned properly.
In the colony counting part of the experiment, the plate provided was of 10
-3
dilution, where the agar
was mostly occupied by a spreader.
Spreaders are colonies of bacteria that spread throughout or on some parts of a petri plate (Hach, 2012).
If spreader colonies occupy less than 50% of the plate, then it must be counted per cm
2
, though if it
occupies more than 50%, meaning it is an excessive spreader, then it must just recorded as a spreader
(McLandsborough, 2005). In this case of the 10
-3
plated dilution, the spreader colony took up more than
half of the plate, meaning it will just be recorded as such.
Spreader colonies, whether bacteria or mold grow in excess moisture in pour plates, though are more
susceptible to grow as surface spreaders in spread plates (Entis, 2002), and are generally of the Proteus
species. Bacteria under the Proteus species are enteric gram-negative rods are aerobes that often
display pleomorphism (the ability to assume distinct forms), are motile, and typically produce swarming
growth on the surface of moist agar plates (Jay, 2000). Given that spreaders love moisture, their growth
can be caused by the condensation of the lid on the petri dish during the cooling process when using the
pour plate method. This is why the plates are inverted during incubation: to avoid condensation from
dropping unto the agar. The incubator may also serve as a source of moisture, as it can provide a humid
environment, then causing the plates to generate moisture. Contamination may also be another cause
of the presence of the spreader, so either the agar was not poured aseptically, or the food itself already
had Proteus spp. present which goes back to improper food handling.

Conclusions
Food and water are needed by all living organisms, so they need to be safe for consumption. If not, then
a person might as well be drinking pollutants or pathogenic bacteria for breakfast, lunch, and dinner,
and for snacks in between.
In order to have food and water safe for people, microbiological methods have been used to determine
whether monsters reside in our food disease-causing bacteria that could lead to great illness or death.
Microorganisms are like air theyre everywhere, at anytime, at anyplace, so nothing is free of them.
They also have two faces, the good and the bad. Good microorganisms give benefits to people, like
probiotics. Bad microorganisms in contrast are those pathogenic bacteria, which is why proper cleaning
is always emphasized everywhere.
References
Caron Ockerman. (November 13, 2012). The debate: Coliforms, Fecal Coliforms and Enterobacteriaceae
as Indicator Organisms. Retrieved October 18, 2014 from http://www.mybiolumix.com/the-
debate-coliforms-fecal-coliforms-and-enterobacteriaceae-as-indicator-organisms/
US EPA (2001) National Primary Drinking Water Standards. Washington, DC: US Environmental
Protection Agency DC
Rompr A., Servais P., Baudart J., de-Roubin MR., Laurent P. (March 2002). Detection and enumeration
of coliforms in drinking water: current methods and emerging approaches. Journal of
Microbiological Methods, 49, pages 31-54.
Houghton, Mifflin, Harcourt (2014).Water Quality Tests. Retrieved October 18, 2014 from
http://www.cliffsnotes.com/sciences/biology/microbiology/aquatic-microbiology/water-quality-
tests
Hach Company. (2012). Heterotrophic Bacteria: Pour Plate Method. Standard Methods for the
Examination of Water and Wastewater. 7th edition.
Archunan, G. (2004). Microbiology. New Delhi: Sarup and Sons.
Food and Drug Administration (FDA). (2013). Revised Guidelines for the assessment of microbiological
quality of processed foods. FDA Circular. Muntinlupa: Filinvest Corporation.
Public Health Agency of Canada. (2011). Proteus spp. Pathogen Safety Data Sheet Infectious
Substances. Canada.
Jay, J., (2000). Modern Food Microbiology. 6
th
ed. Gaithersburg: Aspen Publishers, Inc.
Entis, P. (2002). Food Microbiology - The Laboratory. Washington, D.C.: Food Processors Institute.
McLandsborough, L. (2005). Food Microbiology Laboratory CRC Series in Contemporary Food
Science. Washington:CRC Press