practice in food microbiology manual btft254iu

ObjectiveEach student should be able to- Describe the different types of culture media and their composition, and give several examplesof what each is used for- Describe the various ways

HCMC INTERNATIONAL UNIVERSITYSCHOOL OF BIOTECHNOLOGY

PRACTICE IN FOOD MICROBIOLOGY

MANUAL BTFT254IU

Instructor: Nguyen Thi Huong GiangTeaching Assistant: Bui Thanh Vy

DEPARTMENT OF FOOD TECHNOLOGY

Ho Chi Minh City, 2024

LAB PROGRESS

1 - Preparation and sterilization of BPW, PCA, LST, BGBB

- Sampling of milk sample with pouring and spreading technique for PCA test

- Sampling of juice sample, culturing to LST, and subculturing to BGBB

2 - Preparation and sterilization of PW, TSI, PCA, DRBC

- Counting PCA

- Streaking PCA

- Estimation of total coliforms with most probable number (MPN) test

- Sampling of grape samples and culturing to DRBC

- Stabbing TSI

- Counting DRBC

- Gram staining (PCA, BGBB)

- Motility examination of Salmonella

- Result analysis questions - 35%

- Practice questions (You will be asked to do any labwork skill that you have learned during thecourse) - 30%

2 Report (50%): (5 types of microorganisms)

Each microorganism will follow the below report structure

①Total aerobic count

- Introduction

- Material and Methodology

- Result & Discussion

- Conclusion

- References

②Coliforms

- Introduction

- Material and Methodology

- Result & Discussion

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; Stirringbar; Micropipette; Beaker 500 ml, 1000 ml; Cylinder 100 ml; Alcohol Burner; Thermostatic waterbath, 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, andsurvival 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)

Figure 1.1a A chemically defined medium

Figure 1.1b A complex (undefined) medium

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

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 nutritionallybacteria 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, enrichmentmedia 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 easilyscreened by making that sugar the only carbon source in the medium On the other hand, selectiveinhibition 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 example,media containing potassium tellurite, sodium azide or thallium acetate (at concentrations of 0.1-0.5g/l) will inhibit the growth of Gram-negative bacteria Media supplemented with penicillin (5-50unit/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 withpenicillin 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, theorganisms will produce characteristic changes or growth patterns that are used foridentification or differentiation

3 Preparation of culture media

Rehydrate powder according to manufacturer’s instructions Before sterilization, ensure ingredientsare completely dissolved, using heat if necessary Normally allow 15-20 cm3medium 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 moltencooled 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 thePetri 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 vesselsclosed 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 Oncemelted, agar can be kept molten in a water bath at 50°C until it is ready to be used Sterile agarplates can be pre-poured and stored in well-sealed plastic bags (media-containing base uppermost toavoid 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 solutionand thereby sterilizes them without heat (ø 0.22 µm)

IV Procedure

1 Prepare solid media

a Plate Count Agar (PCA) (Total aerobic count)

- 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

- 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 fromaccumulating on the agar surfaces

b Dichloran Rose Bengal Chloramphenicol Agar (DRBC Agar) - Yeast/Mold determination

- 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 chloramphenicolselective supplement

- Mix well and pour into sterile Petri plates

- When the plates are cool (agar solidified), invert them to prevent condensing moisture fromaccumulating on the agar surfaces

2 Prepare liquid medium

a Lauryl Tryptose Broth (LST) – selective enrichment medium - Coliform determination

- 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

- 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 bubblesafter sterilization)

b Brilliant Green Bile Broth (BGBB) - Coliform determination

- 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 bubblesafter sterilization)

3 Prepare diluent

a Buffered Peptone Water (BPW) (total aerobic, coliform)

- Suspend 20 g in 1000 ml of distilled water (heat if necessary to dissolve the mediumcompletely)

- Sterilize by autoclaving at 15 lb pressure (121oC) for 15 min

- Mix well and dispense into sterile flasks as desired

b Peptone Water (PW) (Yeast/ mold)

- 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, carefullyopen 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, fromhttp://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.) TheMcGraw−Hill

https://courses.lumenlearning.com/microbiology/chapter/how-microbes-grow/

6 Sandle, T (2014, June 18) Assessment of Culture Media in Pharmaceutical Microbiology

https://www.americanpharmaceuticalreview.com/Featured-Articles/163589-Assessment-of-Culture-Media-in-Pharmaceutical-Microbiology/

LABORATORY 2 Sampling, sample preparation, and culture technique

I Objective

Each student should be able to

- Collect samples as representative for the entire lot

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)

1 Samples must be representative for the entire lot

- Sampling is to collect food samples that are representative and then to ensure that changes incomposition 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 errorsmay 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 theproduct) has been mixed with, normally, a nine-fold quantity of diluent

Figure 2.1 The relationship of the operation involved in sampling and analysis

2 Collecting samples for analysis

● 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 shakepowders immediately before testing as the environment may become contaminated by dustparticles

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

It is also possible to homogenize the whole laboratory sample to allow the sampling of ahomogenized test sample It may be necessary to mince or to grind the laboratory sample In thiscase, 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 externalsurface of the package should be disinfected by wiping with alcohol prior to opening or with 2500ppm 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

● Disodium hydrogen phosphate.12H2O 9 g

● Monopotassium hydrogen phosphate 1.5 g

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 bacteriayou started with In bacteriological work, dilutions are usually performed in series of 1/10 or 1/100dilutions 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

4 Culture technique

- Spread plate technique

Figure 2.3 Spread plate technique

- 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 (Total aerobic count).

- Mix the milk sample thoroughly so that the microorganisms are distributed as evenly aspossible by rapidly inverting the sample container 25 times in 30 cm arc within 7 s (avoidfoaming)

- 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, 10-4of milk:

● Take 1 ml of the initial suspension and add it to the next 9 ml tube of BPW to make dilution

10-2

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

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

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

● Flame the neck of molten PCA bottle

● Pour the molten agar into the plate Pour enough agar to cover approximately two-third ofthe bottom of the plate (20ml)

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

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

● 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 (making duplicate petri dishes

for each dilution)

● Vortex the 10-2 dilution tube and dispense 0.1 ml onto the center of the surface of an agarplate

● Dip the bent end of the spreader into the alcohol jar, remove the spreader, and pass itquickly through the flame to allow the remaining alcohol to catch the fire Open the platecover partially and spread the culture onto the agar surface using the sterilized spreader

● Rotate the plate during the spreading to ensure an even distribution of the culture Spreadevenly 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 10-3and 10-4dilutions

● Invert the plates and incubate at 37oC for 24 h

- Streaking

● Label the bottom outer edge of plates as in the pour plating (making duplicate petri dishes)

● 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 theculture (Don’t forget to flame the neck of culture bottle after uncapping and beforerecapping 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 theprimary 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

- 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 agarplate