Total Coliforms, Fecal

Coliforms, & Vibrio

Colera
Hiba Khaled Al Kurdi
ID: 202100288
Instructor: Dr. Malak/ Dr. Lama
Fall Semester 2022-2023
 What are Total Coliforms?
 Total coliform group includes a broad diversity in terms of genus and species, whether or
not they belong to the Enterobacteriaceae family. They are typically present in the
digestive tracks and wastes of worm-blooded animals (including humans) as well as in
environments, such as soil or vegetation.
 Most definitions of coliforms are essentially based on common biochemical
characteristics: they are basically rod-shaped Gram-negative, non-spore-forming Bacilli,
classified as either motile or non-motile; where they both have β-galactosidase in order
to form acids and gases at their ideal growth temperature (35 to 37 °C). They could also
be facultative aerobes or aerobes.
 Total coliform is a large collection of different kinds of bacteria. Fecal coliform is a type
of total coliform that exist in feces. E. coli is a subgroup of fecal coliform.
 How to Detect Coliforms?
Total coliforms are mainly extracted from water samples for lab examinations. This class
of bacteria could be detected by several techniques, either molecular or
microbiological.

1. Briefly, PCR is a molecular method that can be used to detect coliform bacteria by
means of signal amplification: DNA sequence coding for the lacZ gene (beta-
galactosidase gene) and the uidA gene (beta-D glucuronidase gene) has been used to
detect total coliforms.
2. Multiple-tube fermentation technique is a microbiological technique that involves a
three-stage sequential procedure where the results are statistically expressed in terms
of Most Probable Number (MPN). These stages are: presumptive stage, confirmed
stage and completed test. The tubes demonstrating a positive test in all three stages
contain coliforms with a most probable number calculated.
PCR method Multiple-tube fermentation technique
3. Membrane Filter (MF) method: (microbiological method) test samples are filtered through
standard filter paper and then transferred to M-endo or LES Endo Agar mediums. Colonies
appear pinkish-red with metallic sheen after 22-24 hours of incubation. These colonies can be
confirmed as coliforms if they are inoculated in Lauryl Tryptose, produce gas, and then
inoculated in BGLB. If there is gas production in the BGLB tubes, the test is positive for the
presence of coliforms. Enumeration of coliforms by membrane filtration is not totally
specific. When MF is associated with m-Endo media containing lactose, atypical colonies
which are dark red, mucoid or nucleated and without a metallic sheen may occasionally
appear. Atypical blue, pink, white or colorless colonies lacking sheen are not considered as TC
by this technique.
4. Fluorescence in situ hybridization (FISH): a molecular technique that uses peptide nucleic acid
(PNA) probes and an array scanner for rapid detection, identification, and enumeration of
coliforms. The test utilizes Cy3-labeled PNA probes complementary to a specific 16S rRNA
sequence of coliform. Samples are filtered and incubated for 5 h, the membrane filters are then
analyzed by fluorescence in situ hybridization and results are visualized with an array scanner
as fluorescent spots representing coliform microcolonies on the membrane filter surface.
Membrane Filter Method Fluorescence in situ hybridization:
 What are Fecal Coliforms?
Fecal coliform bacteria are a thermotolerent subgroup of the total coliform
bacteria that can be found in the intestines and feces of warm blooded
animals. They possess the same biochemical characteristics of all
coliforms. E. coli is an example that typically resides in the intestinal tract
and thus in the animal's feces.
 How to Detect Fecal Coliforms?

The most preferable technique for determining fecal coliforms is membrane filtration.
Testing samples are extracted from water and are run through a membrane filter with a
specific pore size. The filter surface retains the microorganisms that are present in the
water. The growth of the desired organisms is promoted when the filter is put in a
sterile petri dish and saturated with an appropriate medium. As the fecal coliform
colonies grow, they produce an acid (by fermenting lactose) that reacts with the aniline
dye in the agar, thus presenting blue colored colonies.
 What is Vibrio Cholerae?
 Vibrio cholerae is a species of Gram-negative, facultative anaerobe and comma-
shaped bacteria. It includes various serogroups (not all are pathogenic.

 This bacteria naturally live in coastal water (brackish or saltwater) where they
attach themselves easily to the chitin-containing shells of crabs, shrimps, and
other shellfish.

 This pathogen transitions from aquatic reservoirs into epidemics in human

populations. However, in order to become pathogenic,
the vibrio must acquire the ability to produce the cholera toxin,
which causes the lethal diarrhea of cholera. The ability to produce
this toxin is transferred to the bacteria by a resourceful parasite,
the CTX bacteriophage.
 How to Detect Vibrio Cholerae?
 Vibrio cholera is traditionally separated from environmental water samples
by membrane filtration relying on enrichment step(s) in alkaline peptone
water(enrichment broth) and plating on selective media(preferably
Thiosulfate citrate bile salts sucrose agar) since it is easy to prepare TCBS
agar which turns green. Overnight growth (18-24hrs) of vibrio cholera will
produce a large slightly flattened yellow colonies with opaque centers due
to the fermentation of sucrose in the medium.
 V.cholerae could also be detected by different molecular techniques such as
PCR, IFAG, FISH and others
 When certain if this vibrio cholerae is of a pathogenic serotype, the
diagnosis can be primarily confirmed by rapid tests such as stool dipsticks
or darkfield microscopy can support the diagnosis in settings where stool
culture is not readily available yet.
 References:
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461827/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824014 /
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671094 /
• http://people.envsci.rutgers.edu/phelps/coliforms.pdf
• https://pubs.rsc.org/en/content/articlelanding/2009/an/b81530
7h
Thank You
Hope you enjoyed!