HCMC INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

FOOD MICROBIOLOGY
LABORATORY MANUAL

Instructor: Nguyen Thi Huong Giang

Teaching Assistant: Nguyen Ha My Duyen

DEPARTMENT OF FOOD TECHNOLOGY

Ho Chi Minh City, 2020

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 CONTENT

Contents
LABORATORY 1 Microbiological Culture Media Preparation and Sterilization ..........................................................3
I. Objective ...........................................................................................................................................................3
II. Material ............................................................................................................................................................3
III. Background .......................................................................................................................................................3
IV. Procedure .........................................................................................................................................................5
V. References ........................................................................................................................................................8
LABORATORY 2 Sampling, sample preparation and culture technique .....................................................................9
I. Objective ...........................................................................................................................................................9
II. Material ............................................................................................................................................................9
III. Background .......................................................................................................................................................9
IV. Procedure .......................................................................................................................................................13
V. References ......................................................................................................................................................17
LABORATORY 3 Subculture method and standard plate count ...............................................................................18
I. Objective .........................................................................................................................................................18
II. Material ..........................................................................................................................................................18
III. Principle ..........................................................................................................................................................18
IV. Procedure .......................................................................................................................................................21
V. References ......................................................................................................................................................22
LABORATORY 4 Biochemical activity of bacteria and Most probable number technique ......................................23
I. Objective .........................................................................................................................................................23
II. Material ..........................................................................................................................................................23
III. Principle ..........................................................................................................................................................23
IV. Procedure .......................................................................................................................................................27
V. References ......................................................................................................................................................27
LABORATORY 5 Bacterial Morphology And Staining ...............................................................................................28
I. Objective .........................................................................................................................................................28
II. Material ..........................................................................................................................................................28
IV. Procedure .......................................................................................................................................................29
V. References ......................................................................................................................................................32

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 LABORATORY 1
Microbiological Culture Media
Preparation and Sterilization
I. Objective
Each student should be able to
- Describe the different types of culture media and their composition, and give several
examples of what each is used for
- Describe the various ways culture tubes are capped
- Describe how to prepare and transfer culture media
- Prepare defined and undefined media, and prepare agar plates
- Describe the concept of sterility
- Describe how various media, supplies, and equipment can be sterilized
- Correctly and safely use the autoclave
II. Material
1. Equipment
Test-tube rack, Screw capped test tube; 2-liter Erlenmeyer flask; Petri dish; Pasteur pipette;
Stirring bar; Micropipette; Beaker 500 ml, 1000 ml; Cylinder 100 ml; Alcohol Burner; Thermostatic
water bath, balance, incubator, autoclave, heating oven, biological safety cabinet, pH meter,
vortex mixer, heat-proof Zetex fabric gloves, weighing paper, aluminum foil
2. Media
- Buffered peptone water (BPW)
- Brilliant Green Bile Lactose Broth 2% (BGBB)
- Chloramphenicol Selective Supplement
- Dichloran Rose Bengal Chloramphenicol Agar (DRBC Agar)
- Lauryl Sulphate Tryptose Broth (LST)
- Peptone water (PW)
- Plate count agar (PCA)
- Rappaport Vassiliadis Soya Broth (RVS) (for TA)
- Xylose Lysine Desoxycholate Agar (XLD) (for TA)
III. Background
1. Culture Media Composition
- A microbiological culture medium is a substance that encourages the growth, support, and
survival of microorganisms.
- Main components:
 Water
 N-source: proteins, peptides, amino acids
 Energy source: carbohydrates, proteins
 Additional growth factors: vitamins, minerals, trace elements, an organic salts
- Culture media can be used in different manners:
 In test-tubes
 In flasks
 In petri dishes
2. Media classification
a. Chemical constituents (defined or complex)
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 Figure 1.1a. A chemically defined medium

Figure 1.1b. A complex (undefined) medium

b. Physical nature (liquid, semi-solid or solid)

1.2a. Liquid medium 1.2b. Solid medium 1.2c. Semi-solid medium

(Source: Wikimedia) (Source: Differencebetween) (Source: Differencebetween)
Figure 1.2. Different types of culture media
- Agar medium (solid): are used for isolating bacteria or for determining the colony
characteristics of the microorganisms.
- Liquid medium: are used for various purposes such as propagation of a large number of
organisms, fermentation studies, and various other tests.
c. Function
 Basal media (non-selective media): may be used for growth (culture) of bacteria that do
not need enrichment of the media. Example: Nutrient broth, nutrient agar and peptone
water.
 Enriched media: Addition of extra nutrients in the form of blood, serum, egg yolk, etc., to
basal medium makes enriched media. Enriched media are used to grow nutritionally
bacteria. Example: Blood agar, chocolate agar.
 Selective and enrichment media: Selective media allow certain types of organisms to
grow and inhibit the growth of other organisms. While selective media are agar based,
enrichment media are liquid in consistency. Both these media serve the same purpose.
The selectivity is accomplished in several ways. Organisms that can utilize a given sugar are easily
screened by making that sugar the only carbon source in the medium. On the other hand,
selective inhibition of some types of microorganisms can be achieved by adding dyes, antibiotics,
salts or specific inhibitors which affect the metabolism or enzyme systems of the organisms. For

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example, media containing potassium tellurite, sodium azide or thallium acetate (at
concentrations of 0.1-0.5 g/l) will inhibit the growth of Gram-negative bacteria. Media
supplemented with penicillin (5-50 unit/ml) or crystal violet (2 mg/l) will inhibit the growth of
Gram-positive bacteria. Tellurite agar, therefore, is used to select for Gram-positive organisms,
and nutrient agar supplemented with penicillin can be used to select for Gram-negative
organisms.
 Indicator (Differential) media: are used to differentiate closely related organisms or
groups of organisms. Owing to the presence of certain dyes or chemicals in the media,
the organisms will produce characteristic changes or growth patterns that are used for
identification or differentiation.
3. Preparation of culture media
Rehydrate powder according to manufacturer’s instructions. Before sterilization, ensure
ingredients are completely dissolved, using heat if necessary. Normally allow 15-20 cm3 medium
per petri dish. Dispense in volumes appropriate for sterilization in the autoclave/pressure cooker.
Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing sterile molten
cooled medium to solidify in a sloped position.
4. Pouring a plate
- Collect one bottle of sterile molten agar from the water bath or after cooling from autoclave
- Hold the bottle in the right hand; remove the cap with the little finger of the left hand
- Flame the neck of the bottle
- Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten agar into
the Petri dish and replace the lid
- Flame the neck of the bottle and replace the cap
- Gently rotate the dish to ensure that the medium covers the plate evenly
- Allow the plate to solidify
5. Storage of media
Store stocks of prepared media at room temperature away from direct sunlight. Media in vessels
closed by cotton wool plugs/plastic caps that are stored for future use will be subject to
evaporation at room temperature; avoid wastage by using screw cap bottles.
Re-melt stored agar media in a boiling water bath, pressure cooker or microwave oven. Once
melted, agar can be kept molten in a water bath at 50°C until it is ready to be used. Sterile agar
plates can be pre-poured and stored in well-sealed plastic bags (media-containing base
uppermost to avoid heavy condensation on lid).
6. Sterilization of Media
There are 3 basic methods to sterilize media.
- Autoclaving: 15 min at 121oC
- Boiling: applied for heat sensitive media since 121oC can damage important components
- Bacteriological filter: physically removes bacteria and larger microorganisms from the
solution and thereby sterilizes them without heat (ø 0.22 µm)
IV. Procedure
1. Prepare solid media
a. Plate Count Agar (PCA)
- Prepare 3 parts of PCA (one for spread plating, one for pour plating and one for streaking)
- Suspend 23.5 g in 1000 ml of distilled water
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- Heat to boiling to dissolve the medium completely
- Sterilize by autoclaving at 15 lb pressure (121oC) for 15 min
- Cool to 45-50oC and pour into sterile Petri plates for spread plating and streaking parts
- When the plates are cool (agar solidified), invert them to prevent condensing moisture from
accumulating on the agar surfaces
b. Dichloran Rose Bengal Chloramphenicol Agar (DRBC Agar)
- Suspend 15.75 g in 500 ml of distilled water

- Heat to boiling to dissolve the medium completely

- Sterilize by autoclaving at 121oC for 15 min
- Cool to 50oC and aseptically add sterile reconstituted contents of 1 vial of chloramphenicol
selective supplement
- Mix well and pour into sterile Petri plates
- When the plates are cool (agar solidified), invert them to prevent condensing moisture from
accumulating on the agar surfaces
2. Prepare liquid medium
a. Lauryl Tryptose Broth (LST) – selective enrichment medium
- Composition
Ingredients Weight (g)
Tryptose 20
Lactose 5
Sodium chloride 5
Dipotassium hydrogen phosphate 2.75
Potassium dihydrogen phosphate 2.75
Sodium lauryl sulphate 0.1
Water 1000 ml
Final pH (at 250C) 6.8 ± 0.2
- Dissolve the different components in the water, by heating if necessary
- Adjust the pH, if necessary, so that after sterilization it is 6.8 ± 0.2 at 25°C
- Dispense 10 ml of the broth into 9 tubes containing Durham tubes

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- Place the tubes in a test-tube rack or basket and place in the autoclave
- Sterilize by autoclaving at 121oC for 15 min (the Durham tubes shall not contain air bubbles
after sterilization)
b. Brilliant Green Bile Broth (BGBB)
- Suspend 40.01 g in 1000 ml of distilled water
- Heat if necessary to dissolve the medium completely

- Dispense the medium in quantities of 10 ml in test tubes containing Durham tubes

- Sterilize by autoclaving at 121oC for 15 min (the Durham tubes shall not contain air bubbles
after sterilization)
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3. Prepare diluent
a. Buffered Peptone Water (BPW)
- Suspend 20 g in 1000 ml of distilled water (heat if necessary to dissolve the medium completely)
- Sterilize by autoclaving at 15 lb pressure (121oC) for 15 min
- Mix well and dispense into sterile flasks as desired
b. Peptone Water (PW)
- Suspend 1 g of dehydrated medium in 1000 ml of distilled water
- Mix well to dissolve the medium completely
- Sterilize by autoclaving at 121oC for 15 min
4. Procedure for autoclaving
- TA will demonstrate the use of the autoclave.
- There will be 2 batches of autoclaving (1st batch: solid media, petri plates, some equipment;
2nd batch: BPW and PW in test tubes, other equipment).
- Load the autoclave with the freshly prepared culture media
- Close and lock the autoclave door
- Set the autoclave time for 15 min or longer and select a slow rate of exhaust
- Make certain that the autoclave temperature is set to 121oC
- Start the autoclave by pushing the start button or twisting the knob to the start position
- When the period of sterilization is completed and the pressure in the chamber reads 0,
carefully open the door and remove the containers, using heat-proof gloves
V. References
1. Burdass, D., Grainger, J., & Hurst, J. (2005). Basic practical microbiology: A manual. Reading,
U.K.: Society for General Microbiology.
2. L. Baert (2008) Food microbiology and analysis Practical work presentation. Gent University.
3. Culture of Microorganisms: 5 Steps. (n.d.). Retrieved May 14, 2020, from
http://www.biologydiscussion.com/microorganisms/culture-microorganisms/culture-of-
microorganisms-5-steps/31361
4. Harley, J., & Prescott, L. M. (2002). Laboratory Exercises in Microbiology (5th ed.). The
McGraw−Hill.
5. How Microbes Grow. (n.d.). Retrieved May 14, 2020, from
https://courses.lumenlearning.com/microbiology/chapter/how-microbes-grow/
6. Sandle, T. (2014, June 18). Assessment of Culture Media in Pharmaceutical Microbiology.
Retrieved from https://www.americanpharmaceuticalreview.com/Featured-
Articles/163589-Assessment-of-Culture-Media-in-Pharmaceutical-Microbiology/

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 LABORATORY 2
Sampling, sample preparation and culture technique
I. Objective
Each student should be able to
- Collect samples as representative for the entire lot
- Do an aseptic sampling
- Do dilution series
II. Material
1. Equipment
Blender, sterile plastic bag, Mortar and pestle; Scissor; Screw capped test tube; Erlen; Petri dish;
Pasteur pipette; alcohol jar; spreader; Micropipette; Beaker 500 ml, 1000 ml; Cylinder 100 ml;
Alcohol Burner; Thermostatic water bath, balance, incubator, autoclave, heating oven, biological
safety cabinet, pH meter, votex mixer.
2. Media
- Sterilized Brilliant Green Bile Broth (BGBB)
- Sterilized Buffered peptone water (BPW)
- Sterilized Dichloran Rose Bengal Chloramphenicol Agar (DRBC Agar)
- Sterilized Lauryl Tryptose Broth (LST)
- Sterilized Peptone water (PW)
- Sterilized Plate count agar (PCA)
3. Sample
- Milk
- Grapes
- Vegetables
III. Background
1. Samples must be representative for the entire lot
- Sampling is to collect food samples that are representative and then to ensure that changes
in composition do not take place between collection and analysis.
- Food can be packed in different ways:
 In batch
 In separate units (bottles, cans, boxes)
Use a table with random numbers to collect.
Figure 2.1 illustrates the different stages in sampling and analysis, indicating where sampling
errors may arise as distinct from analytical errors.
- Laboratory sample: sample prepared for sending to the laboratory and intended for
inspection or testing.
- Test sample: sample prepared from the laboratory sample according to the procedure
specified in the method of test and from which test portions are taken.
- Test portion: measured (volume or mass) representative sample taken from the laboratory
sample for use in the preparation of the initial suspension.
- Initial suspension: primary dilution suspension, solution or emulsion obtained after a
weighed or measured quantity of the product under examination (or of the test sample
prepared from the product) has been mixed with, normally, a nine-fold quantity of diluent.

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 Figure 2.1. The relationship of the operation involved in sampling and analysis
2. Collecting samples for analysis
a. Aseptic sampling
Aseptic sampling is a technique used to prevent contamination by the sampling method. Aseptic
sampling involves the use of sterile sampling implements and containers.
There are some methods of material sterilization to be used:
- Dry Heating
 Red heat: flaming until it glows red (metals: inoculation wires)
 Flaming: scalpels, spoons, scissors … immerse in ethanol and immerse in ethanol and
flame
 Hot air: glass material (glass flasks, test tubes, pipettes …) in 2 h at 160oC – 170oC
- Moist Heating
 Boiling in water for 5-10 min: vegetative cells are killed, but spores can survive!!
 Steaming at atmospheric pressure (for sensitive media components)
 Autoclaving: 15-30 min at 121oC using 1 atm overpressure
- Using pre-sterilized plastic tools
- Ethylene oxide gas: 50oC, RH=30%
- Radiation: plastic bags, Petri dishes
- Ultraviolet radiation: air and surfaces
- Filtration: liquid
b. Preparation of sample for analysis
Homogeneous samples including powders and free flowing liquids and concentrates should be
mixed well before removing a portion for testing (for example, by shaking 25 times). Do not shake
powders immediately before testing as the environment may become contaminated by dust
particles.
For heterogeneous products (which contain pieces of different foods), sampling should be
carried out by taking aliquots of each component representative of their proportions in the initial
product.

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It is also possible to homogenize the whole laboratory sample to allow the sampling of a
homogenized test sample. It may be necessary to mince or to grind the laboratory sample. In this
case, to avoid an excessive rise in temperature, do not mince or grind for more than 1 min.
Products stored frozen should be brought to a consistency that allows sampling (i.e. storing at
ambient temperature for a maximum of 3 h or at 2oC ± 2oC for a maximum of 24 h. Samples
should be tested as soon as possible after that.
Packaged products should be opened aseptically, and if necessary and wearing safety glasses
and gloves (if the package cannot be opened without risk of external contamination), the external
surface of the package should be disinfected by wiping with alcohol prior to opening or with 2500
ppm of hypochlorite solution if spore forming bacteria are being sought in the sample.
3. Dilution series
a. Diluent
- Buffered Peptone Water:
 Casein enzymic hydrolysate (peptone) 10 g
 Sodium chlorite 5g
 Disodium hydrogen phosphate.12H2O 9g
 Monopotassium hydrogen phosphate 1.5 g
 Final pH (at 25oC) 7.0 ± 0.2
b. Dilution series
A serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a
more usable concentration. Each dilution will reduce the concentration of bacteria by a specific
amount. So, by calculating the total dilution over the entire series, it is possible to know how
many bacteria you started with. In bacteriological work, dilutions are usually performed in series
of 1/10 or 1/100 dilutions. A 1/10 dilution consists of a 1 ml volume of sample added to 9 ml
volume of diluent, 11 ml volume of sample to 99 ml volume of diluent, or 25 g of food sample to
225 ml diluent.
1 𝑚𝑙 1
= = 1𝑥10−1 = 10−1
9 𝑚𝑙 + 1 𝑚𝑙 10
11 𝑚𝑙 11
= = 10−1
99 𝑚𝑙 + 11 𝑚𝑙 110
25 𝑔 25 1
= = = 10−1
225 𝑚𝑙 + 25 𝑔 250 10

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 Figure 2.2. A serial dilution
When calculating the total dilution, the sample volume is added to the diluent volume. Also note
that when solid foods are used, it is assumed that 1 g of food is equivalent to a volume of 1 ml.
4. Culture technique
- Spread plate technique

Figure 2.3. Spread plate technique

- Streak-Plate Technique

Figure 2.4. Streak plate technique

- Pour-plate technique

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 Figure 2.5. Pour plate technique
IV. Procedure
- Sampling in laminar flow
- Clean & disinfect surfaces
- Clean hand & nails
- Never touch sterile surfaces & materials
- Prepare an alcohol burner during sampling

1. Using aseptic technique to do a sampling of milk product and make a dilution series (PCA
counting).
- Mix the milk sample thoroughly so that the microorganisms are distributed as evenly as
possible by rapidly inverting the sample container 25 times in 30 cm arc within 7 s (avoid
foaming).
- The interval between mixing and removing the test portion shall not exceed 3 min.
- Flame the milk container’s neck.
- Take 1 ml of milk with a sterile pipette and add a 9 ml of BPW

- Shake this primary dilution using a vortex mixer for 5-10 s

- Using separate sterile pipets, prepare decimal dilutions of 10-2, 10-3, 10-4 of milk:
 Take 1 ml of the initial suspension and add it to the next 9 ml tube of BPW to make dilution
10-2

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  Vortex dilution 10-2 for 5-10 s to obtain a 10–2 dilution

 Continue further dilution to obtain 10-3 and 10-4 dilution

- Pour plating
 Label the bottom outer edge of sterile empty plates with your name, the date, the medium,
the organism, and the incubation temperature (making duplicate petri dishes for each
dilution). Also include the dilution factor and plate technique.

 Vortex the 10-2 dilution tube and dispense 1 ml into the bottom haft of the labeled Petri
plate

 Flame the neck of molten PCA bottle

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  Pour the molten agar into the plate. Pour enough agar to cover approximately two-third
of the bottom of the plate

 Mix each plate well by moving it five times in a vertical, clockwise, horizontal and
anticlockwise direction as shown, then allow the plates to set

 Repeat using the 10-3, 10-4 dilutions

 Allow agar to solidify before moving or inverting the plates
 Invert the plates and incubate the plates at 37oC for 24 h
- Spread plating
 Label the bottom outer edge of plates as in the pour plating
 Vortex the 10-2 dilution tube and dispense 0.1 ml onto the center of the surface of an agar
plate
 Dip the bent end of the spreader into the alcohol jar, remove the spreader, and pass it
quickly through the flame to allow the remaining alcohol to catch the fire. Open the plate
cover partially and spread the culture onto the agar surface using the sterilized spreader

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  Rotate the plate during the spreading to ensure an even distribution of the culture.
Spread evenly by making sure the spreader reaches the edges as well as the center of
plate
 Alcohol dip the used spreader and flame the excess alcohol
 Repeat using the 10-3 and 10-4 dilutions
 Invert the plates and incubate at 37oC for 24 h
- Streaking
 Label the bottom outer edge of plates as in the pour plating
 Complete a four-phase streak of the non-diluted culture on the agar plate
 Vortex the culture. Flame the inoculating loop and allow it to air cool. Dip the loop into
the culture (Don’t forget to flame the neck of culture bottle after uncapping and before
recapping the bottle)
 Beginning at the outer edge of the agar, move the loop in a parallel pattern. Flame the
loop
 Do the secondary streak by taking the flamed and cooled loop and crossing back into the
primary streak 2-3 times to pick up organism and then paralleling to spread the organism
 Repeat for the tertiary and the final streak
 Replace the plate lid, invert the plate and incubate at 37oC for 24 h
2. Using aseptic technique to do a sampling of grapes and make a dilution series (Yeast/mold
determination)
- Cut grapes into pieces with a sterilized scissor
- Weigh 25 g of grapes pieces in a sterilized bag

- Add 225 ml PW

- Homogenize sample in the sterilized blender

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- Prepare 3 decimal dilutions of 10-2, 10-3, 10-4
- Label the bottom outer edge of sterilized DRBC plates with your name, the date, the medium,
the organism, and the incubation temperature (making duplicate petri dishes for each
dilution). Also include the dilution factor and plate technique
- Using spreading technique to inoculate each dilution (in duplicate) of the culture to DRBC agar plate
- Incubate the prepared plates aerobically, lids uppermost, in an upright position in the
incubator at 25 ± 1oC for 3 days (It is recommended to incubate the dishes in an open plastic
bag in order not to contaminate the incubator in the event of dissemination of the molds out
of the dishes)
3. Using aseptic technique to do a sampling of vegetables and make a dilution series
(Enumeration of Coliform)
- Cut vegetables into pieces with a sterilized scissor
- Weigh 25 g of vegetables pieces in a sterilized bag
- Add 225 ml PW
- Homogenize sample in the sterilized blender
- Prepare 2 decimal dilutions of 10-2, 10-3
- Use sterile pipette to transfer 1 ml of each dilution into tube containing 10 ml LST (making
triplicate tubes for each dilution)
- Incubate at 30oC or 37oC for 24 ± 2 h
V. References
1. Andrews, W. H., & Hammack, T. S. (2003). Bacteriological Analytical Manual Chapter 1 Food
Sampling and Preparation of Sample Homogenate. Retrieved from -
https://www.fda.gov/food/laboratory-methods-food/bam-food-samplingpreparation-
sample-homogenate
2. BS EN ISO 6887-1:2017. Microbiology of food chain – Preparation of test samples, initial
suspension and decimal dilutions for microbiology examination - Part 1: General rule for
preparation of the initial suspension and decimal dilutions. British Standard Institution.
3. Food Safety And Standards Authority Of India Ministry Of Health And Family Welfare
Government Of India. (2016). Manual on general guidelines on sampling. New Delhi.
4. Greenfield, H., & Southgate, D. A. T. (2003). Food composition data. Retrieved from
http://www.fao.org/3/y4705e/y4705e10.htm
5. L. Baert (2008) Food microbiology and analysis Practical work presentation. Gent University.
6. McLandsborough, & Ann, L. (2005). Food Microbiology Laboratory. CRC Press.
7. Public Health England. (2014). Preparation of samples and dilutions, plating and sub-culture.
Microbiology Services Food Water and Environmental Microbiology Standard Method (1st
ed.). London.
8. Yousef, A. E., & Carlstrom, C. (2003). Food microbiology: A laboratory manual. Hoboken, NJ:
John Wiley & Sons.

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 LABORATORY 3
Subculture method and standard plate count

I. Objective
- To grow and maintain bacterial cultures, to examine cultures for purity or morphology, or to
determine the number of viable organisms
- To estimate the total bacterial count in samples with plate count method
II. Material
- Colony counter
- Incubated Lauryl Tryptose Broth (LST)
- Incubated Plate Count Agar (PCA)
- Isolated Salmonella on Nutrient Agar (NA) media
- Sterilized Triple Sugar Iron (TSI) agar
- Sterilized Brilliant Green Bile Lactose Broth 2% (BGBB)
III. Principle
1. Subculture method
- Subculture of liquid media onto a solid or liquid medium

Figure 3.1. Subculture technique from liquid media to solid media

- Subculture from a solid medium to a liquid medium
- Subculture from a solid medium to a solid medium
2. Slant culture and stab culture

Figure 3.2. Preparation of stab and slant.

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3. The plate count method
A colony-forming unit (CFU) is a unit used to estimate the number of viable bacteria cells in a
sample.
𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒐𝒍𝒐𝒏𝒊𝒆𝒔 (𝑪𝑭𝑼𝒔) = #𝒐𝒇𝒃𝒂𝒄𝒕𝒆𝒓𝒊𝒂/𝒎𝒍 𝒐𝒓 𝒈
The assumption is that each viable bacterial cell is separate from all others and will develop into
a single discrete colony (CFU). Thus, the number of colonies should give the number of live
bacteria that can grow under the incubation conditions employed.
The standard plate count method consists of diluting a sample with sterile saline or phosphate
buffer diluent until the bacteria are dilute enough to count accurately.
Count the colonies with the help of the magnifying glass of a colony counter to facilitate
visualization. Use a colony counter with a 1 cm2 square grid background to serve as a counting
guide (figure 4.1).

Figure 3.3. Colony counter

To carry out this process accurately and rapidly, the analysts should mark the counted colonies,
on the bottom of the plate, with a marker pen to make sure that the same colonies are not
counted repeatedly.
Knowing the dilution factor, volume plated, and number of colonies on the plate (or average from
duplicate plates), count of microorganisms in food is calculated using the following equation:
𝐶𝐹𝑈 𝐶𝐹𝑈 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑓𝑟𝑜𝑚 𝑑𝑢𝑝𝑙𝑖𝑐𝑎𝑡𝑒 𝑝𝑙𝑎𝑡𝑒𝑠
𝐶𝑜𝑢𝑛𝑡 ( 𝑜𝑟 )= (𝐸𝑞. 3.1)
𝑚𝑙 𝑔 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑
For ease of calculation, 1 ml of diluted food sample is considered equivalent to 1 g.
Counting rules
Round off the calculated result to two significant figures. When doing this, if the third figure is
less than 5, do not modify the preceding figure; if the third figure is greater than or equal to 5,
increase the preceding figure by one unit.
Express the result preferably as a number between 1.0 and 9.9 multiplied by the appropriate
power of 10, or a whole number with two significant figures.
Rule 1 – colonies in the range of 20 – 200
- If only 1 plate with a colony count in the range of 20 – 200, calculate the number of colonies
in that plate instead of an average using Eq.3.1.
- If plates from two consecutive dilutions yield 20 – 200 colonies, compute the number of
colonies for each of the two dilutions. If they are not appreciably different, average the
number. If they are substantially different (e.g. the higher CFU/ml is twice the lower one),
report only the lower computed CFU/ml or CFU/g.
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Rule 2 – plate with 1 – 20 colonies
Apply Eq.3.1 to calculate the lowest dilution and report the number as “estimated” or “est.”
Rule 3 – plates with no colonies
Calculate the result of one colony and report as “less than the value obtained”
Figure 3.4. shows example of calculations using rule 1 – rule 3.

Figure 3.4. Example of rule 1 – rule 3 to count colony

Rule 4 – Plate with greater than 200 colonies

Figure 3.5. Counting colonies in crowded plates

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If plates with greater than 200 colonies, using colony counter with gridlines that are 1 cm apart
assists in choosing a representative area of the plate to count.
If there are fewer than 10 colonies/cm2, then count 13 squares (7 consecutive horizontally and 6
consecutive vertically (figure 3.5). The sum of the 13 squares multiplied by 5 equals the estimated
count per 65-cm2 plate.
If there are more than 10 colonies/cm2, count 4 representative squares and multiply the average
by 65 to give the estimated count per plate.
If there are more than 100 colonies/cm2, record the count as > 6.5 x 103 CFU/plate.
Never report the final count in the food sample as “too numerous to count” (TNTC).
IV. Procedure
1. Prepare TSI agar slant
- Suspend 64.52 g in 1000 ml of distilled water
- Heat to boiling to dissolve the medium completely
- Mix well and distribute into 5 test tubes (5- 6 ml/tube), 1 control, 2 typical and 2 atypical
- Sterilize by autoclaving at 121°C for 15 min. Allow the medium to set in sloped form with a
butt about 2.5 cm long

2. PCA counting
Count the number of colonies on Petri dishes (spread plating and pour plating).

Sample:
Colonies
Dilution
Petri dish 1 Petri dish 2
10-2
10-3
10-4
3. Sub-culturing incubated LST into tube containing BGBB (Enumeration of Coliform)

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- Observe the incubated LST tubes, if no gas is formed, re-incubate for further 24 h
- If there is gas and opacity (cloudiness), inoculate with a loopful of incubated LST into tube
containing BGBB

Before incubation After incubation (gas and turbidity)

- Incubate in the incubator at 30oC or 37oC for 24 ± 2 h, or if gas formation is not observed in
this stage, continue incubation for another 24 ± 2 h
4. Streaking the agar slant surface and stabbing the butt (Salmonella confirmation)
- Sterilize inoculating needle, pick 1 well-isolated Salmonella colony on the Nutrient Agar
plate and first STABBING through the center of medium to about 2cm from bottom

- Then STREAKING on the surface of agar slant

- Leave cap loosely and incubate the tubes at 35°C for 18- 24 h
V. References
1. Nguyen, G. T. H. (2019). Food Microbiology Analysis Laboratory Manual. Ho Chi Minh:
International University.
2. Yousef, A. E., & Carlstrom, C. (2003). Food microbiology: A laboratory manual. Hoboken, NJ:
John Wiley & Sons.

22
 LABORATORY 4
Biochemical activity of bacteria and Most probable number technique
I. Objective
Each student should be able to:
- Determine the presence of coliform bacteria in food
- Obtain some index as to the possible number of coliform bacteria present in food
- Understand the biochemical reactions involved in the triple sugar iron agar test
II. Material
- Incubated BGBB
- Incubated TSI
III. Principle
1. Biochemical activity of bacteria – Carbohydrate fermentation - Triple Sugar Iron Agar Test
TSI agar slants contain a 1% concentration of lactose and sucrose, and a 0.1% glucose
concentration. The pH indicator, phenol red, is also incorporated into the medium to detect acid
production from carbohydrate fermentation.
 Interpretation
- Organisms that ferment glucose produce a variety of acids, turning the color of the medium
from red to yellow.
- More amount of acids are liberated in butt (fermentation) than in the slant (respiration).
- Growing bacteria also form alkaline products from the oxidative decarboxylation of peptone
and these alkaline products neutralize the large amounts of acid present in the butt  the
appearance of an alkaline (red) slant and an acid (yellow) butt after incubation indicates that
the organism is a glucose fermenter but is unable to ferment lactose and/or sucrose.
- Bacteria that ferment lactose or sucrose (or both), in addition to glucose, produce large
amounts of acid enables no reversion of pH in that region and thus bacteria exhibit an acid
slant and acid butt.
- Gas production (CO2) is detected by the presence of cracks or bubbles in the medium, when
the accumulated gas escapes.
- If H2S is produced, the black color of ferrous sulfide is seen.

Figure 4.1. Triple Sugar Iron Agar Test Results

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2. Most probable number technique (MPN)
The Most Probable Number technique (MPN) is a quantitative method of analysis that allows
determining the MPN of the target microorganism(s) in a sample, this test involves a multiple
series of Durham fermentation tubes and is divided into two parts: the presumptive and
confirmed tests (Figure 4.2).
Presumtive
Confirmed

Figure 4.2. The Most Probable Number (MPN) procedure

In the presumptive test, dilutions from the sample are added to lactose or LST fermentation
tubes. After 24 to 48 h of incubation at 35oC, one looks for bacteria capable of fermenting lactose
with gas production. In the confirmed test, one transfers material from the highest dilution of
those lactose broth tubes that showed growth and gas production into brilliant green lactose bile
broth, which is selective and differential for coliforms. The tube is incubated for 48 ± 3 h at 35 oC.
Gas formation in the Durham tube is a confirmed test for total target microorganism.
Interpretation:
a. Detection
A tube in which gas formation and opacity (growth) are observed after 24 ± 2 h or 48 ± 2 h is
considered as a positive tube.
b. Enumeration
For each dilution, count the total number of tubes in which gas formation is observed in positive
tubes after 24 ± 2 h and (if used) 48 ± 2 h.
 Calculation using the MPN table
Table 4.1 presents the correlation between inoculated dilutions and the conversion factor to be
applied to the result when the aliquots are different from those tabulated in the MPN tables.
24
 Combination of dilution Conversion factor for the result in the Table
10-1 – 10-2 – 10-3 Read directly
10-2 – 10-3 – 10-4 multiply by 10
10-3 – 10-4 – 10-5 multiply by 100
Table 4.1. Guide for the use of the MPN tables. (Modified from Microbiological Examination
Methods Of Food And Water. A Laboratory Manual (Silva et al., 2013).
In addition to the estimate of the MPN/g or ml of the sample, the MPN tables also present, for
each positive negative tube combination, the upper limit and the lower limit of the MPN
estimate, at the 95% level of confidence. Table 4.2 presents the results of a series of three tubes
inoculated with aliquots of 0.1, 0.01 and 0.001 g or ml of sample and Table 4.3 presents the
results of a series of five tubes inoculated with the same aliquots.

Table 4.1. Most Probable Number (MPN) and 95 percent confidence intervals for three tubes
each at 10-1, 10-2, and 10-3 g or ml inocula. Source: Microbiological Examination Methods Of
Food And Water. A Laboratory Manual (Silva et al., 2013).
The rules for calculating the results:
Rule 1. If 3 inoculated dilutions correspond to the tabulated aliquots (10-1, 10-2, and 10-3), the
result is read directly in the row that corresponds to the combination of positive tubes obtained.
Rule 2. If 3 inoculated dilutions do not correspond to the tabulated aliquots, the result
read in the row that corresponds to the combination of positive tubes should be converted
by applying the conversion factor of Table 4.1.
Rule 3. If more than three dilutions were inoculated, rules one and two apply, but only three
consecutive dilutions are considered.
3.a. If more than 1 dilution presented all tubes positive, the combination considered should
contain the greatest dilution presenting all tubes positive and the two consecutive dilutions.
3.b. If none of the dilutions presented all tubes positive, select the first three consecutive
dilutions for which the middle dilution contains the positive tubes.

25
3.c. If a positive tube occurs in a higher dilution than the three selected, add the number of
positive tubes in this dilution to the highest dilution of the three selected.
3.d. If all dilutions present all tubes positive, select the three highest dilutions.

Table 4.2. Most Probable Number (MPN) and 95 percent confidence intervals for five tubes
each at 10-1, 10-2, and 10-3 g or ml inocula. Source: Microbiological Examination Methods Of
Food And Water. A Laboratory Manual (Silva et al., 2013).

26
Example:

 Calculating using the Thomas formula

𝑃
𝑀𝑃𝑁 (𝑝𝑒𝑟 𝑔 𝑜𝑟 𝑚𝑙) =
√𝑁𝑇
P = Number of positive tubes
N = Sum of the sample quantity inoculated in the negative tubes
T = Sum of the sample quantity inoculated in all tubes
Example: Consider the aliquots and the combination of positive and negative tubes in the series
of three tubes below:
Aliquot 0.1 g 0.01 g 0.001 g
No of positive tubes in 3 0 2 0
P=2
N = (3 × 0.1) + (1 × 0.01) + (3 × 0.001) = 0.313 g
T = (3 × 0.1) + (3 × 0.01) + (3 × 0.001) = 0.333 g
𝑀𝑃𝑁 2
= = 6.2
𝑔 √0.313 × 0.333
IV. Procedure
1. Interpret the TSI test for Salmonella confirmation.
2. Verification of Coliform’s presence and calculation of the most probable number
Observe if there is gas production (positive) and calculate the positive tubes.
V. References
1. Harley, J., & Prescott, L. M. (2002). Laboratory Exercises in Microbiology (5th ed.). The
McGraw−Hill.
2. Nguyen, H. V. H. (2019). Food Microbiology Analysis Laboratory Manual. Ho Chi Minh:
International University.
3. Silva, N. D., Taniwaki, M. H., Junqueira, V.C. A., Silveira, N.F.A., Nascimento, M.D.S.D., &
Gomes, R.A.R. (2013). Microbiological Examination Methods Of Food And Water. A
Laboratory Manual (2nd ed., Vol. 1). London, UK: aylor & Francis Group.
4. Triple Sugar Iron Agar (TSI): Principle, Procedure and Interpretation. Retrieved August 3th,
2020 from https://microbeonline.com/triple-sugar-iron-agar-tsi-principle-procedure-and-
interpretation/
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 LABORATORY 5
BACTERIAL MORPHOLOGY AND STAINING

I. Objective
Each student should be able to
- Learn the proper procedure for preparing a bacterial smear
- Do several simple staining procedures
II. Material
- Microscope
- Clean microscope slides
- Bibulous paper
- Tryptic soy broth or agar slants of Salmonella enterica and Streptococcus salivarius
- Mixture of Salmonella enterica and Streptococcus salivarius in nutrient agar
- Gram staining kit
- Inoculating loop and needle
- Sterile distilled water
- Bunsen burner
- Loeffler’s alkaline methylene blue
- Crystal violet (1% aqueous solution)
- Ziehl’s carbolfuchsin
- Wax pencil
- Immersion oil
- Lens paper and lens cleaner
- Slide holder or clothespin
- Slide warmer
III. Background
1. Bacterial smear
A Bacterial smear is a dried preparation of bacterial cells on a glass slide. In a bacterial smear that
has been properly processed, (1) the bacteria are evenly spread out on the slide in such a
concentration that they are adequately separated from one another, (2) the bacteria are not
washed off the slide during staining, and (3) bacterial form is not distorted.
2. Staining
The use of a single stain or dye to create contrast between the bacteria and the background is
referred to as simple staining.
Basic dyes such as crystal violet (20 to 30 seconds staining time), carbolfuchsin (5 to 10 seconds
staining time), or methylene blue (1 minute staining time) are often used. Once bacteria have
been properly stained, it is usually an easy matter to discern their overall shape.
The most common shapes are presented in figure 5.1.

28
 Figure 5.1. Common Bacterial Shapes
3. Bacterial Motility
Many bacteria show no motion and are termed nonmotile. However, in an aqueous
environment, these same bacteria appear to be moving erratically. This erratic movement is due
to Brownian movement.
Brownian movement results from the random motion of the water molecules bombarding the
bacteria and causing them to move.
True motility (self-propulsion) has been recognized in other bacteria and involves several
different mechanisms. Bacteria that possess flagella exhibit flagellar motion. Helical-shaped
spirochetes have axial fibrils (modified flagella that wrap around the bacterium) that form axial
filaments. These spirochetes move in a corkscrew- and bending-type motion. Other bacteria
simply slide over moist surfaces in a form of gliding motion.
The above types of motility or nonmotility can be observed over a long period in a hanging drop
slide.
IV. Procedure
1. Smear Preparation
- With the wax pencil, mark the name of the bacterial culture in the far left corner on each of
slides.

29
For the broth culture, shake the culture
tube and, with an inoculating loop,
aseptically transfer 1 to 2 loopfuls of
bacteria to the center of the slide.
When preparing a smear from a slant or
plate, place a loopful of water in the
center of the slide. Then aseptically pick
up a very small amount of culture and From solid medium From liquid medium
mix into the drop of water.

- Spread this out to about 1 cm area.

From solid medium From liquid medium

- Allow the slide to air dry at room

temperature
- After the smear is dry, the next step
is to attach the bacteria to the slide by
heat-fixing.

2. Simple staining

Figure 5.2. Simple Staining Procedure

- Cover the smear with methylene blue and allow the dye to remain in the smear for
approximately one minute.
- Using distilled water wash bottle, gently wash off the excess methylene blue from the slide
by directing a gentle stream of water over the surface of the slide.
- Wash off any stain that got on the bottom of the slide as well.
- Saturate the smear again but this time with Iodine. Iodine will set the stain
- Wash of any excess iodine with gently running tap water. Rinse thoroughly.
- Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper
towel.
- Place the stained smear on the microscope stage smear side up and focus the smear using
the 10X objective.
- Choose an area of the smear in which the cells are well spread in a monolayer. Center the
area to be studied, apply immersion oil directly to the smear, and focus the smear under oil
with the 100X objective.

30
3. Gram stain
- Prepare heat-fixed smears of Salmonella
enterica and Streptococcus salivarius and the
mixture of them

- Flood the smears with crystal violet and

let stand for 30 seconds

- Rinse with water for 5 seconds

- Cover with Gram’s iodine mordant and let

stand for 1 minute

- Rinse with water for 5 seconds

- Decolorize with 95% ethanol for 15 to 30

seconds. Do not decolorize too long. Add the
decolorizer drop by drop until the crystal
violet fails to wash from the slide

- Rinse with water for 5 seconds

- Counterstain with safranin and wait for

about 30 seconds to 1 minute

- Rinse with water for 5 seconds

- Blot dry with bibulous paper and examine

under oil immersion

31
 - Gram-positive organisms stain blue to
purple; gram-negative organisms stain pink
to red

4. The Hanging Drop Slide

- With a toothpick, spread a small ring of Vaseline around the concavity of a depression slide
(figure 5.3a). Do not use too much Vaseline.

Figure 5.3. Preparation of a Hanging Drop Slide

- After thoroughly mixing one of the cultures, use the inoculating loop to aseptically place a
small drop of one of the bacterial suspensions in the center of a coverslip (figure 5.2b).
- Lower the depression slide, with the concavity facing down, onto the coverslip so that the
drop protrudes into the center of the concavity of the slide (figure 5.3c). Press gently to form
a seal.
- Turn the hanging drop slide over (figure 5.3d) and place on the stage of the microscope so
that the drop is over the light hole.
- Examine the drop by first locating its edge under low power and focusing on the drop. Switch
to the high-dry objective and then, using immersion oil, to the 90 to 100× objective. In order
to see the bacteria clearly, close the diaphragm as much as possible for increased contrast.
Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between
motility and Brownian movement.
- Discard your coverslips and any contaminated slides in a container with disinfectant solution.
V. References
Harley, J., & Prescott, L. M. (2002). Laboratory Exercises in Microbiology (5th ed.). The
McGraw−Hill.

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