Microbial Analysis

1. Sample preparation:
➢ Sampling:
Sampling is a critical step in microbial analysis, involving the collection of food samples from
processing lines, storage houses, or markets.

Sample:
▪ The sample should be representative of the entire food lot.
▪ It should be collected randomly to avoid bias by employing various sterile tools
like pipettes, stirrers, swabs, scissors, knives.

Sampling device:
Sampling devices differ based on the nature of the food, with liquid and small particles
sampled using tubes, spoons, and droppers, while large materials like cheese are cut with a
knife.
▪ Sterile spoon pipette, stirrer, swab etc. are used for taking samples.
▪ Sterile scissors, knife, can opener etc. are used for opening food packages.
▪ Liquid and small particles are sampled with sampling tube, spoon, droppers etc.
▪ 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.

Number of samples:
▪ The number of samples varies; for Salmonella testing, co-samples are collected,
while five samples are needed for other organisms.

➢ Techniques for sample collection:

Techniques for sample collection differ for big and small containers, involving cleaning,
sterile cutting instruments, and agitation for homogenous mixture in liquid samples.
o When the product is in big container: Steps in sample collection are:
▪ Outer surface of container is cleaned by washing or by 70% ethanol.
▪ Container is opened with the help of sterile cutting instrument.
▪ If the food is in bulk, the sample are collected from various places within
container.
▪ In case of liquid sample, it is first agitated to form homogenous mixture and
sample is taken.
o When the food is in small container:
 ▪ When the sample is in small container, they should be taken directly to the
lab for analysis.
➢ Preparation of sample:
▪ For solid food samples: Solid food is mixed with sterile diluent in mechanical
blender to obtain homogenous suspension. For example, when 10gm food is added
to 100ml diluent, 10-1 dilution is obtained.
▪ For liquid food samples: Serial dilution is performed to obtain 10-1, 10-2, and 10-3
dilutions
➢ Transportation of sample:
▪ Transportation precautions include preventing microbial multiplication and death,
delivering samples to the lab immediately, and cooling perishable samples to 0-5°C,
using insulated containers.
▪ Frozen products must be maintained in frozen condition until analysis, and sampling
reports should accompany the samples for documentation purposes.

2. Media preparation
To isolate, identify and to study the characteristics of microorganisms it is essential to grow
them. Microorganisms required various nutrients for their growth. In addition to nutrients they
require moisture, proper degree of temperature, proper type of gaseous environment and a
suitable pH. The environment from which an organism can get all these requirements is called
culture medium.

Culture media are principally used to cultivate microorganisms. Now ever for special suppose
they may be modified by altering their composition or by adding certain necessary substances.
Liquid and Solid Media:
Microbial culture media may be provided either as a broth or a solid gel. The liquid media may
be converted to solid media by addition of suitable amount of gelling agent (or solidifying
agent). The most commonly used solidifying agent in bacteriology is agar-agar a
polysaccharide obtained from a marine algae. It has the very useful property of gelling at
approximately 450C and liquefying at 98 C. Thus, once it gelled the medium remains solid
through the temperature growth range of about, all microorganisms when liquefied. The
medium remains liquid until cooled to within the heat tolerance of almost all microorganisms.
This facilitates us to use a solid medium for cultivating organisms. Agar has the additional
useful property of being resistant to hydrolysis by most microorganisms.

As organisms grow in liquid media (or both) it utilizes components of the medium and excretes
by products of bacterial metabolism into the medium. Provided the medium is originally free
of these by products. Use can be made of their production to help identify the organisms.
Number of such biochemical reaction use broth media. Besides organisms when they grow in
a broth they exhibit a typical growth pattern, which also helps in their identification. Thus,
growth media are largely used in study of a pure culture.
As stated above liquid media are solidified by addition of agar. When grown in solid media,
organisms remain confined to suite of inoculation. As they grow there it results in production
as a large mass of cells, which becomes visible to naked eye such a mass, is called a colony.
The appearance of these colonies is often typical of the tissues. This makes possible the
isolation of the single species of bacteria from a mixed population.

Preparation of culture media essentially involves the following steps:

1. To dissolve the ingredients in a specified amount of distilled water. If any of the

ingredients is heat labile then it should be added at the end after sterilization of medium.
2. Adjustment of pH of the medium to the desired value.
3. If the medium to be prepared is a liquid medium they it should be distributed in a
specified amount into suitable type of containers (test tubes, flask) and the mouth of the
container is plugged with cotton wool. If the medium to be prepared is a solid media
then suitable amount of agar is added to the medium and then the medium is heated to
dissolve agar. Then the medium is dispersed in flask and mouth of the flask is plugged
with a cotton wool.
4. A paper wrapped around the mouth of the container and then the medium is sterilized.
5. Broth can be directly used for inoculation when cooled. Solid media are poured in
sterile containers (petridish or test tubes) under aseptic conditions. They are allowed to
cool I solidify.

Media, which are used for general cultivation of bacteria, are nutrient broth and nutrient agar.
Both these media are no synthetic. They are designed to support the growth of a wide range of
bacteria and consist of peptone, meat extract and sodium chloride and distilled water at a pH
of approximately 7.4. Agar is the surplus ingredient in nutrient agar medium. Peptone is a
source of nitrogen. It also acts as a buffer due to its amphoteric nature. Meats extract supplies
a wide range of growth factors including mineral salts and amine acids. Sodium chloride
maintains the osmotic pressure. Agar-agar present in nutrients agar acts as a solidifying agent.

Following are media which are generally used in bacteriology

1) Nutrient agar:
Composition: Peptone 1 gm
Meat extracts 0.3 gm
NaCl 0.5 gm
D/W 100 ml
pH 7.6
Agar powder 3 gm

Note: Dissolve all the ingredients except agar powder and then adjust the pH to 7.6.
Add agar powder and heat to dissolve. Autoclave it at 15 lbs pressure for 15 minutes.
 Use: It is used for general cultivation and isolation of bacteria.

2) Nutrient broth:
Composition: Peptone 1 gm
Meat extracts 0.3 gm
NaCl 0.5 gm
D/W 100 ml
pH 7.6

Autoclave at 15 lbs pressure for 15 minutes.

Use: It is used for general cultivation of bacteria.

3) MacConkey’s agar:
Composition: Peptone 2 gm
NaCl 0.5 gm
Bile salt 0.5 gm
Lactose 1 gm
D/W 100 ml
pH 7.6
Agar powder 2.5 gm

Autoclave at 15 lbs pressure for 15 minutes. Add 1 ml of 1% Neutral red solution before
pouring plates. The medium contains bile salt the surface-active agents. It inhibits growth
of gram-positive bacteria. Thus, the medium is a selective medium for growth of gram-
negative bacteria. The medium also contains lactose and a pH indicator dye neutral red. If
bacteria are able to ferment lactose it results in production of acid. Due to acid production
the indicator neutral red gives pink color. Thus, the colony of the lactose non-fermenting
bacteria is colorless. Thus, the medium also acts as a differential medium for lactose
fermenting and non- fermenting bacteria.

Use:
1. For selective cultivation of gram-negative bacteria, especially coli form.
2. For differentiation of lactose fermenting and lactose non-fermenting bacteria.

4) Eosin methylene blue agar:

Composition: Peptone 10 gm
Lactose 10 gm
K2HPO4 5 gm
D/W 1 liter
pH 7.6
Agar powder 2.6 gm
1% Eosin yellow 20 ml
0.5% Methylene blue 12ml
 Autoclave at 15 lbs pressure for 15 minutes. Then add separately sterilized 10% solution
of lactose so as to give final concentration of 0.2%. Add 20 ml of Eosin yellow and 15 ml
of 0.5% Methylene blue and pour into the plate. It differentiates between lactose fermenter
organisms and lactose non- fermenter organisms. The dyes, eosin and Methylene blue
inhibit growth of gram-positive organisms. The medium is also useful in differentiation of
e.g.,E. coli and Enterobacteraerogens. E. coli which is a strong fermenting organism
produces large amount of acids. The dyes eosin and Methylene blue gets precipitated due
to heavy acid production due to this, colonies of E. coli are having greenish metallic sheen
and are dark centered. E. aerogens though ferments lactose, produces lesser amount of
acid which is not enough to precipitate dye, Greenish metallic sheen is thus not observed
in case of E. aerogens. Instead, the colonies are purplish pink in color and are mucoid. The
colonies of lactose non-fermenting organisms are colorless.
Use:
1. Used to differentiate lactose fermenter organisms from lactose non-fermenting
organisms.
2. Used to differentiate E. coli and E. aerogens.

5) Potato dextrose agar:

Potato dextrose agar is a selective medium used for the cultivation of yeasts and molds.
The use of potato extract promotes growth of yeast and molds and the low pH (3.5) helps
to inhibit the growth of bacteria while favoring the growth of many yeasts and molds.

Materials:
1. Test tubes and flasks
2. Raw potatoes, dextrose, agar, sterile tartaric acid (10%)
3. Other materials as indicated for nutrient broth/agar

Procedure:
Composition:
Potato : 200 g
Dextrose : 20 g
Agar : 20 g
Water : 1000 ml
Final pH : 3.5 (after sterilization)
1. Peel the potatoes and cut into small cubes.
2. Take 200 g of potato cubes in 500 ml of water and steam for 1hr.
3. Mash by stirring well.
4. Strain through fine muslin and make up the filtrate to 1000ml.
5. Add dextrose and agar; dissolve the agar by boiling it.
6. Dispense the medium into test tubes and flask and plug them with cotton.
7. Sterilize at 121°C, 15 psi pressure for 20 min.
8. Adjustment of pH to 3.5 should be made at the time of pouring the plates.
3. Aerobic Plate Count:
The Aerobic Plate Count (APC) provides an estimation of viable microorganisms in given test
samples or products. Test results determine the number of aerobic mesophilic bacteria.

Aerobic Plate Count is also known by other names such as standard plate count, aerobic
mesophilic count, total plate count, or aerobic colony count. Aerobic plate count method’s
limitation is that it does not distinguish between different bacterial colonies on a given sample.

Importance of Aerobic Plate Count:

▪ Aerobic Plate Count test is primarily used to check microbiological quality of test
samples to avoid public health concerns caused by microbial contamination. The
results of the test indicate the major hygiene issues, quality of raw material, storage
conditions, and shelf life of the product or anticipated changes that occur due to enzyme
degradation.
▪ Aside from this, the Aerobic Plate Count Test method can also be used in the field of
agriculture and to monitor the quality of drinking water.

Procedure:

▪ Test sample are prepared based on the type of food products (frozen, chilled, precooked,
or prepared foods) or cosmetics products (Liquids, Solids and powders, cream and oil-
based products, Aerosol of powders, soap, liquids, and other materials, Anhydrous
materials).
▪ Homogenized solution containing the test sample is serially diluted 10 times
▪ (10-1 to 10-10).
▪ 0.1 ml of each dilution is transferred and plated on different petri dishes containing agar
growth medium.
▪ Plates are incubated at 35°C ± 1°C for 48 ± 2 hours.
▪ After incubation, the plates are examined for the presence of aerobic bacteria.

4. Yeast and mold count:

Yeast cells generally surpass the size of most bacteria, exhibiting considerable variation in
dimensions, ranging from 1 to 5 micrometers in width and 5 to 30 micrometers or more in
length. While typically egg-shaped, some yeast cells can be elongated or spherical, each species
possessing its distinct characteristic shape. Even within a pure culture, individual cell size and
shape vary significantly based on factors such as age and environment. Notably, yeasts lack
flagella or other locomotive organelles.
In molds, the thallus comprises two fundamental components: the mycelium and spores, which
are resistant, dormant cells. The mycelium forms an intricate network of filaments known as
hyphae. Hyphae typically emerge from a germinating spore, extending germ tubes that elongate
and branch, ultimately forming hyphae.

Hyphae, each measuring about 5 to 10 micrometers in width, stand in stark contrast to bacterial
cells, which are usually only 1 micrometer in diameter. Structurally, hyphae consist of an outer
tube-like wall surrounding a filled or lined cavity called the lumen. The wall is composed of
microfibrils primarily made up of hemicelluloses or chitin. Growth occurs distally near the tip,
with the major elongation region just behind it. Young hyphae may develop cross walls through
centripetal invagination, forming incomplete septa with central pores allowing for
protoplasmic streaming. Hyphae come in three forms: non-septate (coenocytic), septate with
uninucleate cells, and septate with multinucleate cells.

Mycelia, the vegetative or reproductive structures of molds, play distinct roles. Vegetative
mycelium penetrates the medium to obtain nutrients, absorbing soluble compounds through the
walls. Reproductive mycelia, responsible for spore production, often extend into the air. The
mycelium of molds can either be a loosely woven network or an organized, compact structure,
as seen in mushrooms.

Note: Potato Dextrose Agar is one of the selective medium used for the cultivation of yeasts
and molds. The use of potato extract promotes the growth of yeasts and molds and the low pH
of the medium helpsyeasts and molds growth and also inhibits growth of bacteria.
Composition of Potato Dextrose Agar (PDA):
▪ Potato: 200g
▪ Dextrose:20g
▪ Agar:20g
▪ Water: 1000ml
▪ Final pH 3.5 ( with 10% sterile tartaric acid)
Sterilize the medium and cool to 45°C. Before adding into Petri dish adjust agar pH to 3.5
with the help ofsterile tartaric acid
Materials:
▪ Food sample
▪ Sterile saline tubes (9ml)
▪ Sterile pipettes (10ml, 1ml and 1.1ml)
▪ Sterile Petri dishes
▪ Water bath (to maintain 45°C)
Procedure:
▪ Take I ml well - mixed sample of raw or pasteurized milk and add to 9 ml phosphate
buffer tube. Mix the contents properly. This makes 1:10 dilution (dilution).
 ▪ From the first dilution tube, take out 1 ml and add to another 9 ml phosphate buffer
tube. This makes 1:100 dilution.
▪ Similarly, make required number of serial dilutions.
▪ Add 1 ml diluted sample from appropriate tubes to sterilized labelled Petri plates (make
duplicate Plates).
▪ Pour 15-20 ml of melted and cooled SPC agar (cool to 45°C) into each of the plates
▪ Mix well and allow the plates to solidify
▪ Incubate the plates at 25 C for 4-5 days along with the control plate in inverted position.
▪ Remove the plates after 4 - 5 days and count colony forming units (CFU) and record
the results in a tabular form.
▪ Multiply cfu with appropriate dilution factor and express the count as cfu/ml of milk
sample.

5. Coliform count
The coliform count is a microbiological measurement used to assess the presence of coliform
bacteria in a given sample, typically water or food. Coliform bacteria, which are naturally found
in the intestines of warm-blooded animals, serve as indicators of potential faecal
contamination. The count is determined through methods such as the most probable number
(MPN) or membrane filtration, focusing on the bacteria's ability to ferment lactose. Elevated
coliform counts in water or food samples may signal a risk of harmful pathogen presence,
emphasizing the importance of monitoring and testing to ensure public health and safety.
Regulatory authorities often establish guidelines for acceptable coliform counts in various
samples, with regular testing playing a crucial role in preventing waterborne or foodborne
diseases.
Materials:
1. Samples of milk
2. VRBA agar
3. Dilution blanks (9ml Phosphate buffer tubes)
4. Dairy Bacteriological Pipettes; 1.1 ml
5. Petri plates (98 mm outer diameter, 15 mm depth)
6. Incubator at 37 °C
7. Colony counter

Procedure:
▪ Take I ml well - mixed sample of raw or pasteurized milk and add to 9 ml phosphate
buffer tube. Mix the contents properly. This makes 1:10 dilution (dilution).
▪ From the first dilution tube, take out 1 ml and add to another 9 ml phosphate buffer
tube. This makes 1:100 dilution.
▪ Similarly, make required number of serial dilutions.
 ▪ Add 1 ml diluted sample from appropriate tubes to sterilized labelled Petri plates (make
duplicate Plates).
▪ Pour 15-20 ml of melted and cooled SPC agar (cool to 45°C) into each of the plates
▪ Mix well and allow the plates to solidify
▪ Add another 5 ml of same agar as cover laver.
▪ Incubate the plates at 37°C for 24 hours along with the control plate in inverted position.
▪ Remove the plates after 24 hours and count dark red colonies measuring > 0.5 mm
diameter as colony forming units (CFU) and record the results in a tabular form.
▪ Multiply cfu with appropriate dilution factor and express the count as cfu/ml of milk
sample.

6. Food pathogens: Salmonella & Shigella

Principle:
The procedure consists of five to six distinct stages. The initial handling of the food and the
non-selective enrichment stage (pre-enrichment) vary according to the type of food examined.
▪ Non- Selective Enrichment (pre-enrichment): The test sample, is initially inoculated
into a non-inhibitory liquid medium to favour the repair and growth of stressed or sub
lethally-injured salmonellae arising from exposure to heat, freezing, desiccation,
preservatives, high osmotic pressure or wide temperature fluctuations
▪ Selective Enrichment: Replicate portions of each pre-enrichment culture are
inoculated into two enrichment media to favour the proliferation of salmonellae through
a selective repression or inhibition of the growth of competing microorganisms

Materials and Special Equipment:

▪ Nutrient broth (NB)
▪ Sample
▪ Tetrathionate broth
▪ Iodine
▪ Incubators

Procedure:
DAY 1:
▪ Prepare 225ml of pre-enrichment media (NB) in 500 ml flask.
▪ Add 25 ml of food sample in pre-enrichment media.
▪ Incubate at37°C for 24 hours at 120 rpm.
DAY 2:
▪ Prepare 2 tubes of 10ml of enrichment media (Tetrathionate broth) and boil it.
▪ Let it cool and add iodine.
▪ Transfer 1 ml from pre-enriched media into enriched media under aseptic conditions.
▪ Incubate at 37°C for 24 hours
DAY 3:
▪ Prepare selective media (XLD/DCA/SSA)
▪ Collect loopful of enriched media and streak on selective media plates
▪ Incubate at 37°C for 24 & 48 hours