PHT 381 Lab# 4. A Culture medium:- ❊ An artificial preparation which contains the essential elements and nutrients needed by the m.o to grow. (most.

PHT 381PHT 381Lab# 4Lab# 4

A Culture medium:-A Culture medium:-

❊ ❊ An An artificial preparationartificial preparation which which contains the contains the essential elements and essential elements and nutrientsnutrients needed by the m.o needed by the m.oto grow. (most bacteria &fungi) to grow. (most bacteria &fungi)

❊❊ Strict intracellular organismsStrict intracellular organisms (e.g., (e.g., some bacteria & all viruses)→ only some bacteria & all viruses)→ only cultures of living eukaryotic cellscultures of living eukaryotic cells..

❊ It may be:• Liquid (broth)• Solid (containing agar) • Semisolid (containing low conc of agar)

Most common ingredients:-

1. Essential elements and nutrients.

2. Solidifying agents.

Inoculation:Inoculation: Culturing of sterile media with m.o Culturing of sterile media with m.o [Inoculation loop].[Inoculation loop].

Incubation:Incubation: Placing the culture into the incubator Placing the culture into the incubator at optimum temperature for growth.at optimum temperature for growth.

Growth:Growth: Multiplication Multiplication (↑number)(↑number) to to quantities sufficient to be quantities sufficient to be seen by seen by naked eye..naked eye..

Bacterial growthBacterial growth in the lab has in the lab has 2 main 2 main forms:forms:

1-1- Development of Development of Colonies Colonies ( the ( the macroscopic products of 20-30 cell macroscopic products of 20-30 cell divisions of a single bacterium on divisions of a single bacterium on solid solid mediamedia))

2-2- Turbidity Turbidity (macroscopic clumps) (macroscopic clumps) of a clear of a clear fluid mediumfluid medium..

Bacterial GrowthBacterial Growth

1- Macroscopical Examination • (colony morphology):• Characters of colonies.• Hemolysis on blood agar.• Pigment production.

2- Microscopical Examination:• Examination of wet mount preparation.• Examination of stained preparation.

3-Biochemical Tests:

(The ability to attack various substances e.g., carbohydrate breakdown;

or to produce particular metabolic products e.g., enzymes.

4-Additional Tests:

such as seriological tests

Colony vs. CellColony vs. Cell

Colonies morphology

(Macroscopical examination)

Cells

(Microcopical examination)

Colony vs. CellColony vs. Cell

Colonies morphology

(Macroscopical examination)Cells

(Microcopical examination

• Contamination:

Introduction of undesirable m.o.

• Asepsis:

Processes designed to prevent m.o. from reaching a protected environment.

• Aseptic technique: Practices used by microbiologists to exclude all organisms from contaminating media or contacting living tissues.

• An aseptic technique must be used when inoculating culture media to:

1- prevent contamination of cultures

2- prevent infection of laboratory workers and enviroment.

Isolation of Pure Colonies of Isolation of Pure Colonies of Microorganism Microorganism

“Streak Plate Method”“Streak Plate Method”

In natural environments, bacteria & other m.o exist in mixed populations.

To study the cultural, morphological, and physiological characteristics of an individual organism, it is essential, first of all, that the organisms are separated from other species

i.e. we must have pure culture of the micro-organism.

““Streak Plate Technique”Streak Plate Technique”

Streak plate method is one of the most frequently used methods of getting a pure culture from a mixed culture.

The individual cells are separated from each other by certain distance on the surface of the agar.

After incubation, each single deposited cell divide many times and finally form visible mass of growth “COLONY”.

The streak Plate MethodThe streak Plate Method

• The culture prepared from a single type of colony is regarded as a pure culture.

• The streak Plate Method is used for:Checking the purity of a bacterial culture.Isolating individual species from a mixture

culture.

The streak Plate MethodThe streak Plate Method

• Objective:- for isolation of individual species of a mixed

broth culture.

• Materials:- Nutrient agar plate.

Mixed broth culture of

Serratia marcescens and Staph. aureus.

The streak Plate MethodThe streak Plate Method

• Procedure:

S & SS & S

Aseptic technique Aseptic technique

Invert the plate and Invert the plate and Incubate for 24h at 37Incubate for 24h at 37℃℃

Drop of the cultureDrop of the cultureFlam & Cool

Flam & Cool

Flam & Cool

The streak Plate MethodThe streak Plate Method

The streak Plate MethodThe streak Plate Method

Description of ColoniesDescription of Colonies

Sources of ContaminationSources of Contamination

• Objective: To identify some of the sources of contamination present in the lab.

✔ in order to avoid them

• Contamination from hands.

• Contamination from breath.

• Contamination from air.

• Contamination from bench.

Sources of ContaminationSources of Contamination

Sterile nutrient agar plate a- unwashed

b- washed with water

c- disinfected with alcohol

d- control incubate the plate Inverted at 37°c for 24

hr. Record the appearance of the plate

a b

c d

1.Contamination from hands:

Sources of ContaminationSources of Contamination

2. Contamination from breath: Take a sterile nutrient agar

plate Hold it in front of your mouth Cough and breath vigorously Invert the plate and incubate

for 1 day at 37 °c Record the appearance of the

plate.

Sources of ContaminationSources of Contamination

3.Contamination from air: Expose one sterile nutrient

agar plate on the bench for 30 min

Invert the plate and incubate for 1 day at 37 °c

Record the colonial appearance

30 min

Sources of ContaminationSources of Contamination

4.Contamination from bench: Take a sterile nutrient agar plate

and mark out 2 sections on its base Take a swab from unclean part of

the bench and press it over one section

Take another swab from cleaned part with disinfectant and press it over the second section

Invert the plate and incubate for 1 day at 37 °c

Record the result.

uc

c

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