Methods of Sterilization

Sterilization refers to the complete destruction of all living organisms, including bacterial spores
and viruses. The word sterile means free from or the absence of all living organisms. Basically
they are 2 types. Physical methods of sterilization comprise moist heat and dry heat. Chemical
methods include gas and liquid solutions.

Chemical Agents: They are 3 types

a. Disinfectants: An agent used to disinfect inanimate objects or surfaces but is generally to

toxic to use on human tissues. Ex: Phenol, Lysol

b. Antiseptica: An agent that kills or inhibits growth of microbes but is safe to use on human
tissue. Ex: Dettol, Iodine,

c. Anti-microbial agents: An agent that kills microorganisms or stops their growth Ex:
antibiotics are used against bacteria and antifungals are used against fungi, Viricides used against
virus.

Physical Sterilization:

Red heat: Platinum or Nichrome wire heated in Bunsen flame.

Steam heat: Autoclave and Arnold steam sterilization are examples for Steam heat type
sterilization.

Dry-heat: Hot-air-oven is the examples.

Radiation: Near ultraviolet, visible light, infrared, microwave, radio waves, and low-frequency
radio frequency (longwave) are all examples of non-ionizing radiation and Gamma rays, X-rays,
and the higher ultraviolet are ionizing radiations

Filtration: Micro filters are generally used for filtration. Cialis filters, Chanberland filters,
Berkefeld filters are examples.
 Preparation of microbiological media (bacterial, algal & fungal)

Preparation of bacterial culture medium

Preparation of agar planets with agar slants

Materials and equipment: distilled water, measuring cylinder, flask, bacteriological chemicals,
laboratory scales, chemical spoons, 1N NaOH solution, 1N HCl solution, pH indicator paper or
pH meter, cotton gloves, sterile, empty Petri dishes, Bunsen burner, autoclave

Practice:

Measure the components of the medium (e.g. TSA or nutrient) into a flask containing 9/10
volume of the solvent. Dissolve the solid components and fill with the remaining solvent up to
final volume. If the medium contains heat sensitive components (like sugars), they must be
separately sterilised in solution (e.g. by filter sterilisation), and then mixed with the already
sterilised and cooled agar medium.

Close the flask with cotton plug and cover with aluminium foil, put into the autoclave and start a
sterilisation cycle (see EXERCISE 1). This cycle could be intermitted when the internal
temperature has reached 121°C, at that temperature every component (e.g. agar-agar) will be
dissolved correctly.

Check the pH of the medium with an indicator paper or with a pH meter and adjust to the proper
value with NaOH or HCl solution.

Pour the 60-70°C medium into the dispenser. Add 5-6 mL medium to each test tube, close them
with caps and place them into a test tube basket.

Place the tubes into the autoclave and complete a whole sterilisation cycle for 20 min at 121°C

Put the test tubes onto a slanting stage to let the medium solidify in the test tubes.

Label the slants according to the type of the medium and perform a sterility test: incubate the test
tubes at 28°C for 24 hours, and check for sterility.

Cool the sterilised medium to 55°C.

Take out the cotton plug and flame the mouth of the flask over a Bunsen burner, and then pour
the medium into sterile, empty Petri dishes (15-20 mL into each Petri dish).

Keep the Petri dishes horizontally until the medium completely solidifies

Label the plates according to the type of the medium and perform a sterility test
Preparation of Nutrient broth

Beef extract 3g
Peptone 5g
Distilled water 1 liter

Heat to dissolve. Dispense 10 ml portions into tubes or 225 ml portions into 500 ml Erlenmeyer
flasks. Autoclave 15 min at 121°C. Final pH, 6.8 to 7.2. Use Non-absorbent cotton.

Preparation of Potato dextrose agar (PDA) for fungal culture

To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30
min.

Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial
dehydrated form).

Mix with Dextrose, Agar and Water and boil to dissolve.

Autoclave 15 min at 121°C.

Dispense 20-25 ml portions into sterile 15 × 100 mm petri dishes.

Final pH, 5.6 ± 0.2.

Pringsheims Medium for Blue Green Algae

Ingredients Gms / Litre

Potassium nitrate 0.200

Magnesium sulphate 0.010

Ammonium hydrogen phosphate 0.020

Calcium chloride 0.005

Iron (II) chloride 0.0005

Suspend 0.24 grams in 1000 ml distilled water. Heat if necessary to dissolve the medium
completely. Dispense and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes

Cultural characteristics observed after an incubation at 25-27°C for 1 week.

 Isolation of bacteria by streak, spread and pour plate methods

A pure culture theoretically contains a single bacterial species. There are a number of procedures
available for the isolation of pure cultures from mixed populations. A pure culture may be
isolated by the use of special media with specific chemical or physical agents that allow the
enrichment or selection of one organism over another. The differential and selective procedures
will be utilized later in this course. Simpler methods for isolation of a pure culture include: (i)
spread plating on solid agar medium with a glass spreader and (ii) streak plating with a loop. The
purpose of spread plating and streak plating is to isolate individual bacterial cells (colony-
forming units) on a nutrient medium.

Both procedures (spread plating and streak plating) require understanding of the aseptic
technique. Asepsis can be defined as the absence of infectious microorganisms. However, the
term is usually applied to any technique designed to keep unwanted microorganisms from
contaminating sterile materials.

Material:

1. Seven 9-ml dilution tubes of sterile saline

2. Seven nutrient agar plates
3. 1.0 ml and 0.1 ml pipets
4. Glass spreader aka hockey stick
5. 95% ethyl alcohol in glass beaker (WARNING: Keep alcohol away from flame!!)
6. Mixed overnight broth culture of Staphylococcus aureus and Serratia marcescens

A. Spread Plate Technique

In this technique, the number of bacteria per unit volume of sample is reduced by serial dilution
before the sample is spread on the surface of an agar plate.

1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix the nutrient
broth tubes before each serial transfer. Transfer 0.1 ml of the final three dilutions (10-5,
10-6, 10-7) to each of three nutrient agar plates, and label the plates
2. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar
plates until the medium no longer appears moist. Return the spreader to the alcohol.
3. 4. Repeat the flaming and spreading for each of the remaining two plates.
4. 5. Invert the three plates and incubate at room temperature until the next lab period.

B. Streak Plate Technique

The streak plating technique isolates individual bacterial cells (colony-forming units) on
the surface of an agar plate using a wire loop. The streaking patterns shown in the figure
below result in continuous dilution of the inoculum to give well separated surface
colonies. Once again, the idea is to obtain isolated colonies after incubation of the plate.
 1. Label two nutrient agar plates No. 1 and No. 2.

2. Prepare two streak plates by following two of the 3 streaking patterns shown in the
figure below. Use the 10-1 dilution as inoculum.

3. Invert the plates and incubate at room temperature until the next lab period

C. Pour plate technique

Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL.
(Follow serial dilution technique)

Inoculate labeled empty petri dish with specified mL (0.1 or 1.0 mL) of diluted specimen

Pouring the molten agar and incubation

1. Collect one bottle of sterile molten agar (containing 15 mL of melted Plate Count Agar or any
other standard culture media) from the water bath (45°C).
Pouring the molten agar medium
2. Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
3. Flame the neck of the bottle.
4. 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 the lid.
5. Flame the neck of the bottle and replace the cap.
6. Gently rotate the dish to mix the culture and the medium thoroughly and to ensure that the
medium covers the plate evenly. Do not slip the agar over the edge of the petri dish.
7. Allow the agar to completely gel without disturbing it, it will take approximately 10 minutes.
8. Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.