Cultivation of baceria
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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

Cultivation of baceria

By

Dr. Emad AbdElhameed Morad

Lecturer of Medical Microbiology and Immunology


Cultivation of baceria

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


Cultivation of baceria

Plating out technique

Dr. Emad AbdElhameed Morad


Cultivation of baceria

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


    Cultivation of baceria

    Liquid media

    • Peptone water:

    • 1% peptone + 0.5%NaCl.

    • Base for indole test.

    • Base for sugar media.

    Composition

    Uses

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Nutrient broth with bacterial growth

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Nutrient agar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    Nutrient agar showing pseudomonas

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    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


    Cultivation of baceria

    Bacterial motility

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Blood agar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    Blood agar showing beta hemolysis

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    Blood agar showing alpha hemolysis

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Chocolate agar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Loffler’s serum

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Lownstein Jensen medium

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    TCBS medium

    TCBS with Vibrio cholerae

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    MacConkey’s agar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    MacConkey’s agar with both lactose and non lactose fermenters

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Triple sugar iron

    A B C D E F G H

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    (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


    Cultivation of baceria

    Quiz

    A:

    B:

    C:

    D:

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    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


    Cultivation of baceria

    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


    Cultivation of baceria

    Cultivation of anaerobes

    Culture media for anaerobes

    Anaerobic jar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Robertson cooked meat medium

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    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


    Cultivation of baceria

    Anaerobic jar

    Dr. Emad AbdElhameed Morad


    Cultivation of baceria

    Thank You

    Dr. Emad AbdElhameed Morad


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