Food and Water Microbiology

WATER AND FOOD SAMPLING FOR

MICROBIAL ANALYISIS: ISOLATION AND
ENUMERATION OF BACTERIA IN WATER AND
FOOD
 Introduction
• A wide range of bacterial, viral and protozoan
diseases result from contamination of water with
human and other animal faecal wastes.
• Isolation and detection of these pathogens can take
several days, weeks, or months
• However, absence of one particular pathogen does
not rule out the presence of another
 Indicator Organism Concept
• These are organisms that serve as an index of possible
water contamination by pathogens.
• The indicator organism should:

– Be suitable for the analysis of all types of water:

tap, river, ground, recreational, sea e.t.c.
– Be present whenever enteric pathogens are present.

– Survive longer than the hardiest enteric pathogen.

Indicator Organism Concept cont…
– The assay procedure for the indicator organism
should have great specificity i.e. other bacteria
should not give positive results.
– The procedure should also have high sensitivity
and detect low levels of the indicator organism.
– The testing method should be easy to perform.
Indicator Organism Concept con…
– Level of indicator organism in the water should
have a relationship to the degree of faecal
pollution.
 Coliforms
• Are members of the Family Enterobacteriaceae
• They are:
– Facultative anaerobic - Grows with or without
oxygen.
– Gram negative
– Non-spore forming
– Rod shaped
– Ferment lactose with gas and acid production
within 48 h at 35 C
 Coliform group
• The Coliform group include:
– Enterobacter aerogenes
– E. coli
– Klebsiella pneumoniae
• Other indicator organisms include fecal
enterococci.
• Mostly used as indicators for brackish and
marine waters.
 Water for bacteriology
Preparation
Chlorinated water - add sodium thiosulphate to neutralize chlorine
(0.5 ml of 10% solution or a small crystal)
Sampling water from Tap/ pump
• remove attachments
• wipe, clean and flame outlet
• allow to flow (at least one minute)
• Sampling water from water course or reservoir - collect from a
depth of at least 20 cm
• Sampling water from dug well - do not allow the bottle to touch
the sides of the well
 Water for bacteriology
Collect atleast 200 ml of water sample from the
source in sterile glass bottles or autoclavable
plastic bottles with tight screw capped lids or
securely fit stoppers/caps.
 Water for bacteriology
Handling and transportation
Test the water sample within 3 hours of collection
• keep at ambient temperature
If delayed:
• Keep sample on ice
• test refrigerated sample within 24 hours
 Testing for Coliforms: MPN
• A three step process is used. This includes:

– Presumptive step

– Confirmed and

– Complete test step

• The presumptive step involves the use of tubes inoculated with three
different sample volumes i.e. to give the most probable number.
• This methods is therefore called the Most Probable Number
method.
• The complete process starting from presumptive,
confirmed and complete test takes 4 days of
incubation and transfers.
• Due to the wide range of bacteria under coliforms,
some of which may not be from the intestinal tract, a
test for faecal coliforms has been developed.
• Faecal coliforms are coliforms derived from the
intestine of warm blooded animals.
• They grow at 44.5oC.
 Description of MPN
• Also called Multiple Tube Fermentation Method

• The water must be serially diluted to extinction

• Multiple tubes (3 - 5) of media are inoculated across t

he increasing series of dilutions.
• The tubes are then incubated at

– 35 oC or

– 44.5 oC
 Description of MPN
• Positive growth tubes are counted.

• Positive tubes are identified by observing change in

colour in the indicator media (e.g. yellow colour in
MacConkey broth which is usually purple).
• Most-Probable-Number (MPN) table is used to
estimate microbial density.
The most probable number method
(MPN)
• Other tests for coliforms and fecal coliforms
include:
– Membrane filtration technique

– The presence-absence (P-A) test for coliforms and

– Colilert defined substrate test for detecting both

coliforms and E. coli.
 Membrane Filtration Technique.
• Common and often preferred
– Measured sample (100 ml) of water is filtered through a
0.45 μM membrane filter.
– All bacteria are “trapped” on the filter surface
– Filter paper is placed on surface of culture medium to
allow for growth of colonies (coliforms).
– The media used should be selective/differential for
pathogens (e.g., E. coli)
– Incubate
• 35 o C total coliform
• 44.5 o C faecal coliform
• Count colonies
• A resuscitation step can be used for chlorine stressed
microorganisms.
Advantages of MFM
• Good reproducibility
• Single step results often possible
• Filters can be transferred between different media
• Large volumes can be processed to increase assay
sensitivity
• Time saving
• Lower total cost in comparision to MPN method.
Disadvantages
– High turbidity waters limit volumes sampled.

– High population of background bacteria cause

overgrowth.
– Metals and phenols can adsorb to filters and inhibit
growth.
 The Presence-Absence Test
• This is a modification of MPN method.

• A large volume of water (100 ml) is incubated in a

single culture bottle with triple strength broth
containing lactose broth, lauryl tryptose broth and
bromocresol purple indicator.
 The Presence-Absence Test
• Assumption: No coliforms should be present in 100
ml of drinking water.
• A positive result is production of an acid (media
changes to yellow color)
• This needs a confirmatory test.
 Related Colilert defined substrate test
• Water sample (100 ml) is added to a specialized
medium containing o-nitrophenyl-ᵦ-D-
galactopyranoside (ONPG) and 4-methylumbelliferyl-
ᵦ-D-glucuronide (MUG) as the only nutrients.
• If the medium turns yellow within 24 hrs at 35 oC, then
coliforms are present causing hydrolysis of ONPG.
• Hydrolysis of ONPG results into the release of o-
nitrophenol.
• The presence of E. coli is confirmed by exposing the
culture under long-wavelength UV light to check for
fluorescence.
• E. coli modifies MUG to produce the fluorescent
product.
 Food samples
Collect suspect food samples earliest
Collect aseptically - sterile tools, containers
Solid Food:
– cut 100-200 grams from the centre with sterile knife
– raw meat or poultry - refrigerate in a sterile plastic jar
Liquids:
– shake to mix, use sterile tube
– water used for cooking -- 1-5 litres
 Food samples
Contact surfaces:
– (utensils and/or equipment) for food processing swab
using moisten swab with sterile 0.1% peptone water or
buffered distilled water; put the swab in an enrichment
broth.
Handling and transportation
– As fast as possible
– Keep perishable foods at 2-8oC
– Cool hot food rapidly - put containers under cold running
water
– Pack samples to prevent spillage
Enumeration Serial dilution and viable
plate count

Figure 6.22
 Enumeration
• To calculate the number of bacteria per mL of diluted
sample one should use the following equation:
•  Number of CFU/mL = Total count/Volume plated (mL)
× total dilution factor used
• For example, if for 1×10-8 dilution plate you plated 0.1
mL of the diluted cell suspension and counted 200
bacteria, then the calculation would be:
•  
• 200/0.1 mL × 10-8 or 200/10-9 or 2.0 × 1011 bacteria
per mL.
 Assignment
If there were 3.0 × 1011/ml of bacterial cells

out of 0.1 plated volume at dilution of 1.0 × 10 -8.

a). How many colonies were in the counted

plate?

b). What was the dilution factor?

 Reference

• J. L. Oblinger and J. A. Koburger. 1975.

Understanding and Teaching the Most Probable
• Number Technique.“
J. Milk Food Technol. 38(9): 540-545