LECTURE 2:

PART B - TECHNIQUES ON
MICROBIOLOGICAL
ANALYSIS OF FOOD

MCB106, 2024-2025
Lynn E. Rallos
 Microbial analysis of food

•Food is a good culture

_____________for
medium growth
of many microorganisms.
•The presence of microorganisms in
food causes decomposition and
spoilage of food.
•Therefore, microbial analysis of food is
important in ________________
quality assurance of food.

* Not all spoilage microbes are pathogenic

Why analyze food microbiologically?

• Meet __________________for
desired specification
raw material, intermediate, and
finished product,
• Identify ________
risk factors,
• Process ___________,
verification and
• Confirm that
_________________are
regulatory guidelines followed.
Microbial analysis of food gives idea
about:

quality
•The ____________ of food

•The ______________________
sanitary condition of food

•The ________________
storage condition

•The __________________________
types of microorganisms in food

•The quality of ____________________

raw material

•Evaluation of __________________
effectiveness
Bottomline: ensure food safety
one of major reference

- published by USDA
- this manual presents FDA's preferred lab
procedures for microbiological analyses of foods and
cosmetics
Guidelines | CODEXALIMENTARIUS FAO-WHO
Books – ICMSF | International Commission on Microbiological Specifications for Foods
Importance of Proper Sampling of Food

❑ Sample or specimen received for testing must be

_____________and
adequate proper condition
in _______________

❑ Because interpretations about a large consignment of food are based

on a relatively small sample of the lot, established sampling
procedures must be applied _______________.
uniformly

❑ A representative
_____________sample is essential when pathogens or toxins
are sparsely distributed within the food or when disposal of a food
shipment depends on the demonstrated bacterial content in relation
to a legal standard.

Lifted from the Bacteriological Analytical Manual

Importance of Proper Sampling of Food

❑ The number of units that comprise a representative

sample from a designated lot of a food product must be
__________________________.
statistically significant
❑ The composition and nature of each lot affect the
____________
homogeneity and _______________of
uniformity the total
sample mass.
❑ The proper statistical _________________,
sampling procedure
according to whether the food is solid, semisolid,
viscous, or liquid, must be determined by the collector at
the time of sampling by using the Investigations
Operation Manual (see BAM)
Lifted from the Bacteriological Analytical Manual
Steps in microbial analysis of food:

1. Sampling:
•Sample of food can be obtained from the
processing line, from storage house, or from market.

•The sample:
• The sample should berepresentative
_______________of whole
lot of food.
• Sample should be collected randomly
_____________ from
the lot.
• The principle is to avoid ___________
bias and to draw
_____________number
sufficient of sample units.
 Steps in microbial analysis of food:

•Sampling device:
• Sterile spoon pipette, stirrer, swab etc.
are used for taking samples
• Sterile scissors, knife, can opener etc.
are used for opening food packages
• Sterile Bacti disc cutter or another suitable
opening device (Terriss Consolidated T-14-
182)
• Sampling tube, spoon, droppers etc.
Liquid and small particles are sampled with
Large food materials (e.g., cheese) are cut by
• knife
For surface samples,
• knife, swabbing or sticky cello phone tape
is used
• Sticky cello phone tape is pressed on the surface of solid
agar to transfer microorganisms
Steps in microbial analysis of food:

•Number of samples:

1.the reason for the test, e.g. ongoing quality

control, compliance with regulations or product
investigations.
2.any statistical analyses that may be required.
3.Kind of sample
4.Kind of test to be used
Steps in microbial analysis of food:

2. Techniques for sample collection:

•When the product is in BIG container: Steps in sample
collection are:
• washing - outer surface of container is cleaned by washing or by 70% ethanol
• Container opened with the help of sterile cutting instrument
if the food is in bulk,
• sample is collected from various places within container.
• In case of liquid sample, agitate then take sample

•When the food is in SMALL container:

• Sample should be taken directly to the lab for analysis.
Steps in microbial analysis of food:

3. Preparation of sample:

•For solid food sample: mixed with sterile diluent in a

mechanical blender (or stomacher) to obtain a
homogenous suspension. eg., when 10g of food is added to 100ml diluent, a
10^-1 dilution is obtained.

•For liquid food sample: serially diluted to obtain a series

of dilutions (e.g. 10-1, 10-2, 10-3 ).

The serial dilution scheme depends on the type of food and the knowledge of the analyzer about
microbiology
Steps in microbial analysis of food:

4. Transportation of sample:

•Multiplication and death of sample (microorganisms)

should be prevented during transport of sample.
•The sample should be delivered to the lab
immediately.
•Perishable and unfrozen sample should be cooled to
0-5oC and transported in insulated container.
•Frozen product must be maintained in frozen
condition until analysis.
•Sampling reports should be prepared and sent along
with sample.
Steps in microbial analysis of food:

5. Sampling report:

The report should provide the following

information?
* Identification of the sample:
* Sampling location and conditions
- sample ID or code
- sampling description
- quantity/volume
- sampling date and time
* Sampling location and conditions
- sampling location
- storage and transport conditions
- environmental conditions
Steps in microbial analysis of food:

6. Sampling plan:

•Choice of sampling plan depends upon the

___________ ___________________,
organism present in food the __________________
nature of food and
whether the food is ________________________________
raw or processed or eaten directly,
_______________________
type of consumer i.e. normal, infant, old person or
ill person.

•Sampling plan is basically a statement of criteria of

_______________________________-
acceptance ________________based
upon examination of the required number of sample
units by a specific method.
•Sampling plan are of two types:

•Two class plan:

• It consists of three specification : 'n', 'c', and 'm'
This plan is simplest and is used to 'accept' or 'reject' food product
e.g. Two class of certain food for coliforms is:
• N=5, c=2 and m=102
• It means 5 sample must be tested.
• In order for that to be acceptable ______________________
not more than two samples
must contain greater than 100 coliforms per gram.
• The food will be _____________,
rejected if 8 or more sample units exceed 100
organism per gram.

e.g. n=5, c=0, m=0 (Salmonella). It means food will be rejected if any one
sample out of five contained organisms.

where, n = number of sample units to be tested

m = maximum number of organisms per gram. value about this is non-acceptable
c =the maximum no. of allowable samples that may extend the
microbiological criteria 'm'.
when this number is exceeded, the lot is rejected
•Sampling plan are of two types:

•Three class plan:

• four specifications : n, c, m, and M
• ____
M the quantity is used to separate marginally
accepted quality from unacceptable quality.
• Number or value above M in any sample is
______________.
unacceptable
eg., n = 5, c = 2, m =100, M = 150 e.g., food A. coliform
it means 5 samples must be tested. if not more than two samples
• 2 samples exceed equal or more than an
organism, it is __________________
acceptable if any one sample.
• Sample contains equal or greater than M, than
sample is __________________________
non-acceptable
Sample procedure for dairy product:

•Milk and milk product:

•No. of samples:
• Fluid milk= 5 field samples
• Dried milk and ice cream = 10 field samples
• Cheese = 5 field samples
• Butter = 5 field sample
•Methods of analysis: ?
* Enumeration of mesophilic aerobic bacteria
* Enumeration of coliforms
* Enumeration of Staphylococcus aureus (cheese)
* Detection of Salmonella
* For butter, enumeration of yeast and mold as well as coliforms
•Mesophilic aerobic bacteria in Milk:
• Raw milk should not exceed total plate count = ______________
2x 10^5/ml
• Pasteurized milk should not exceed total plate count = ___________
2 x 10^4/ml
• At 37oC, coliform = _______________
0 (best)
• At 42oC, fecal coliform = __________
0 (best)

•For dried milk,

• Mesophilic aerophilic bacteria = n= 5, c= 2 , m=5x10^4, M =5x10^5
• Coliform= n= 5 , c= 2 , m= 2 , M= 10^2
• Staphylococcus aureus = n= 5 , c= 1 , m= 10 , M= 10^2
• Salmonella = n= 10, c= 0 , m= 0
Sampling procedure for meat and meat
products:

•No. of field samples=5

•Method of analysis
• Enumeration of mesophilic aerobic
bacteria
• Detection of Salmonella
Sampling procedure for meat and meat
products:

Microbial analysis of Carcass sampling (raw meat):

•Sterile large swab moistened in 0.1% peptone water
•carcass wiped with the swab
•carcass again cleaned with dry pad
•pads are taken and 250ml of diluent are added in the
pad to disperse the microorganisms from the trap.
•Serial dilution
•Plate count at 20oC.
•For detection of Salmonella, plating is done in a
selective medium.
 Example

Compaore et al. PLOSOne, 2022, Microbiological quality assessment of five common foods sold at different
points of sale in Burkina-Faso - PubMed (nih.gov)
Objective of the research

The aim of the present study was to assess the microbial

quality of five ready-to-eat food such as bread, pasta, rice
with sauce, beans and milk sold in five localities of Burkina
Faso namely, Ouagadougou, Bobo-Dioulasso, Dakola,
Cinkansé and Niangoloko.
 Table 1. Subtotal of ready-to-eat food samples.

Compaore MKA, Kpoda SD, Bazie RBS, Ouedraogo M, Valian M, et al. (2022) Microbiological quality assessment of five common foods sold at
different points of sale in Burkina-Faso. PLOS ONE 17(4): e0258435. https://doi.org/10.1371/journal.pone.0258435
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0258435
How did they do their sampling

One hundred and one samples divided into five groups including
15 bread samples, 12 for beans samples, 12 pasta samples, 19
rice with sauce samples and 43 milk powder samples were
collected from November to December 2021 in five localities of
Burkina Faso. Samples were purchased at local stores markets,
street food and restaurants. Sampling sites include ordinary
restaurants, local markets and shops.

The concerned localities are Ouagadougou, Bobo-Dioulasso,

Dakola, Cinkansé and Niangoloko. All samples were taken
aseptically in sterile plastic bag, kept in an insulated cold box
containing ice boxes or stored at room temperatures. Samples
reached the laboratory are immediately analyzed or kept under 4°C
until used.
Analysis parameters

Parameters applied to each group of samples were those

recommended by the Codex Alimentarius for cooked foods
namely:
Total aerobic mesophilic flora
Coliforms
Thermotolerant coliforms
Yeast and molds
Escherichia coli
Salmonella spp.
Coagulase-positive staphylococci.
Anaerobic Sulfito Reductive (ASR) bacteria (milk)
Clostridium perfringens (milk)
Bacillus cereus were (milk).