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Cultivation of baceria. By. Dr. Emad AbdElhameed Morad. Lecturer of Medical Microbiology and Immunology. INOCULATION PROCEDURE. P lating out technique: Aims at spreading out the bacteria on the surface of the medium. Each bacterium will divide to give a separate colony.

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Cultivation of baceria

By

Dr. Emad AbdElhameed Morad

Lecturer of Medical Microbiology and Immunology

slide2
INOCULATION PROCEDURE
  • Plating out technique:
  • Aims at spreading out the bacteria on the surface of the medium.
  • Each bacterium will divide to give a separate colony.
  • The loop is sterilized and loaded with the bacterial mixture present in the culture or the specimen. Then, it is smeared over area A to give the main inoculum.
  • The loop is then sterilized and after cooling, drawn into three parallel lines B across a fresh part of the medium.
  • The procedure is repeated as shown in the figure.
  • The plate is incubated at 37 degree for 24 hours.

Dr. Emad AbdElhameed Morad

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Plating out technique

Dr. Emad AbdElhameed Morad

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CULTURE MEDIA
  • Liquid media.
  • Solid media.
      • Simple media
      • Enriched media
      • Selective media
      • Differential (indicator media)
  • Sugar media.
  • Enrichment media.
  • Cultivation of anaerobic bacteria (culture media & anaerobic jar)

Dr. Emad AbdElhameed Morad

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Liquid media
  • Peptone water:
  • 1% peptone + 0.5%NaCl.
  • Base for indole test.
  • Base for sugar media.

Composition

Uses

Dr. Emad AbdElhameed Morad

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Composition
  • Nutrient broth (meat infusion broth):
  • Meat extract + 1% peptone + 0.5% NaCl.
  • pH is adjusted to 7.2 and sterilized in the autoclave.
  • Base for nutrient agar medium.

Uses

Dr. Emad AbdElhameed Morad

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Nutrient broth with bacterial growth

Dr. Emad AbdElhameed Morad

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Simple

Solid media

  • Nutrient agar:
  • Nutrient broth + 2 – 3% agar.
  • Agar is sea weeds that is dissolved by heating at 100 degree and become s solidified at 45 degree.
  • Base for blood agar.
  • Detect exopigment production by pseudomonas.

Composition

Uses

Dr. Emad AbdElhameed Morad

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Nutrient agar

Dr. Emad AbdElhameed Morad

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Nutrient agar showing pseudomonas

Dr. Emad AbdElhameed Morad

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Composition
  • Nutrient slope agar:
  • Nutrient broth + 2– 3% agar.
  • Poured in tubes. It has a sloping surface.
  • Preservation of bacteria.

Uses

Dr. Emad AbdElhameed Morad

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Composition
  • Deep soft agar:
  • Nutrient broth + 0.5 – 1% agar.
  • Poured in tubes. It has horizontal upper surface.
  • Detection of bacterial motility.
  • Cultivation of anaerobes.

Uses

Dr. Emad AbdElhameed Morad

slide13
Bacterial motility

Dr. Emad AbdElhameed Morad

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Enriched

Solid media

  • Blood agar:
  • Nutrient agar + 10% sheep or horse blood.
  • The sterile blood is added to the nutrient agar at 55 degree to avoid hemolysis of red blood cells.
  • Base for chocolate agar.
  • Differentiates between bacteria according to the type of hemolysis produced: beta, alpha and gamma hemolysis.

Composition

Uses

Dr. Emad AbdElhameed Morad

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Blood agar

Dr. Emad AbdElhameed Morad

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Blood agar showing beta hemolysis

Dr. Emad AbdElhameed Morad

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Blood agar showing alpha hemolysis

Dr. Emad AbdElhameed Morad

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Composition
  • Chocolate agar:
  • The same as blood agar.
  • But, the temperature of the medium is raised to 100 degree. During heating, red blood cells become ruptured with release of hematin.
  • Cultivation of highly fastidious bacteria as neisseria and hemophilus.

Uses

Dr. Emad AbdElhameed Morad

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Chocolate agar

Dr. Emad AbdElhameed Morad

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Composition
  • Loffler’ serum:
  • 3 parts horse serum + 1 part glucose broth.
  • Serum is the solidifying agent.
  • Cultivation of diphtheria bacilli.

Uses

Dr. Emad AbdElhameed Morad

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Loffler’s serum

Dr. Emad AbdElhameed Morad

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Selective

Solid media

  • Lowenstein-Jensen medium:
  • Beaten eggs + mineral salts + malachite green.
  • Malachite green inhibits bacteria other than tubercle bacilli.
  • Cultivation of tubercle bacilli.

Composition

Uses

Dr. Emad AbdElhameed Morad

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Lownstein Jensen medium

Dr. Emad AbdElhameed Morad

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Composition
  • TCBS medium:
  • Thiosulphate, citrate, bile as selective agents.
  • Sucrose as test sugar.
  • Thymol blue and bromothymol blue as pH indicators.
  • Cultivation of Vibrio cholerae.
  • Vibrio cholerae grow s as yellow colonies on TCBS.

Uses

Dr. Emad AbdElhameed Morad

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TCBS medium

TCBS with Vibrio cholerae

Dr. Emad AbdElhameed Morad

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Differential

Solid media

  • MacConkey’s medium:
  • Peptone : nutrient.
  • Sodium taurocholate: inhibits non intestinal bacteria.
  • Lactose + neutral red indicator.
  • Differentiates enterobacteriacae into:
    • lactose fermenters which give rose pink colonies.
    • Non lactose fermenters which give pale colonies.

Uses

Dr. Emad AbdElhameed Morad

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MacConkey’s agar

Dr. Emad AbdElhameed Morad

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MacConkey’s agar with both lactose and non lactose fermenters

Dr. Emad AbdElhameed Morad

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Composition
  • Triple sugar iron (TSI) agar:
  • Glucose 0.1%, Lactose 1%, sucrose 1% are the test sugars.
  • Phenol red is the pH indicator.
  • Ferrous sulphate to detect H2 S production.
  • 0.5% agar (soft agar cracks on gas production).
  • Differentiates between:
  • Bacteria according to their effect on sugars and production of H2S.

Uses

Dr. Emad AbdElhameed Morad

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Triple sugar iron

A B C D E F G H

Dr. Emad AbdElhameed Morad

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(From left to right)
  • Uninoculated control.
  • Red slant and red butt, no black color= no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced.
  • Red slant and black butt = no lactose or sucrose fermentation, H2S has been produced.
  • Red slant with yellow butt = no lactose or sucrose fermentation, glucose is fermented, no H2S has been produced.
  • Yellow slant, yellow butt and black coloration = Lactose, sucrose and glucose fermented, and H2S has been produced.

F, G. Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration = Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced.

H. Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas been.

Dr. Emad AbdElhameed Morad

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Quiz

A:

B:

C:

D:

Dr. Emad AbdElhameed Morad

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Sugar media
  • Six tubes.
  • Each tube contains peptone water + specific sugar in a concentration 1%.
  • The sugars are glucose, lactose, maltose, mannite, sucrose, salicin.
  • Each tube contains also Andrade’s indicator which changes to pink if acid is produced.
  • An inverted small tube (Durham’s tube) is immersed in the medium and gas production is revealed by collection of bubbles at its apex.

Composition

Dr. Emad AbdElhameed Morad

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Uses
  • Differentiate between bacteria according to their fermentative effect on different sugars and whether the fermentation is accompanied by production of gas or not.

Fermentation of glucose, maltose, mannite with acid only

Fermentation of all sugars with acid + gas

Dr. Emad AbdElhameed Morad

slide35
Enrichment media
  • These are fluid media that contain substances that enhance the growth of some bacteria and not others.
  • Examples:
  • Selenite broth: an enrichment medium used for isolation of salmonella and shigella.
  • Tetrathionate broth: an enrichment medium used for isoaltion of salmonella.

Dr. Emad AbdElhameed Morad

slide36
Cultivation of anaerobes

Culture media for anaerobes

Anaerobic jar

Dr. Emad AbdElhameed Morad

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Culture media for anaerobes
  • Examples of anaerobic bacteria: clostridia, bacteriodes, lactobacilli.
  • Meat contains hematin, glutathione and lipids which reduce the oxygen in the medium.

Robertson cooked meat medium

Thioglycolate broth

Deep soft agar

Dr. Emad AbdElhameed Morad

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Robertson cooked meat medium

Dr. Emad AbdElhameed Morad

slide39
Anaerobic jar
  • It is a plastic jar with tightly fitted lid.
  • Hydrogen is introduced from hydrogen generator envelope.
  • Hydrogen reacts with the O2 in the jar in the presence of a catalyst suspended from the lid forming water.
  • Anaerobic indicator such as methylene blue is placed in the jar. It is colorless under anaerobic conditions and changes into blue in the presence of O2.

Dr. Emad AbdElhameed Morad

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Anaerobic jar

Dr. Emad AbdElhameed Morad

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Thank You

Dr. Emad AbdElhameed Morad

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