Cultivation of baceria By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology

INOCULATION PROCEDURE Dr. Emad AbdElhameed Morad 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. INOCULATION PROCEDURE Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Plating out technique Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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) CULTURE MEDIA Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Peptone water: 1% peptone + 0.5%NaCl. Base for indole test. Base for sugar media. Liquid media Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Nutrient broth with bacterial growth Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Simple Solid media Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Nutrient agar Dr. Emad AbdElhameed Morad 12/1/2018

Nutrient agar showing pseudomonas Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Nutrient slope agar: Nutrient broth + 2– 3% agar. Poured in tubes. It has a sloping surface. Preservation of bacteria. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Deep soft agar: Nutrient broth + 0.5 – 1% agar. Poured in tubes. It has horizontal upper surface. Detection of bacterial motility. Cultivation of anaerobes. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Bacterial motility Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Enriched Solid media Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Blood agar Dr. Emad AbdElhameed Morad 12/1/2018

Blood agar showing beta hemolysis Dr. Emad AbdElhameed Morad 12/1/2018

Blood agar showing alpha hemolysis Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Chocolate agar Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Loffler’ serum: 3 parts horse serum + 1 part glucose broth. Serum is the solidifying agent. Cultivation of diphtheria bacilli. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Loffler’s serum Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Lowenstein-Jensen medium: Beaten eggs + mineral salts + malachite green. Malachite green inhibits bacteria other than tubercle bacilli. Cultivation of tubercle bacilli. Selective Solid media Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Lownstein Jensen medium Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

TCBS with Vibrio cholerae Dr. Emad AbdElhameed Morad TCBS medium TCBS with Vibrio cholerae Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Differential Solid media Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad MacConkey’s agar Dr. Emad AbdElhameed Morad 12/1/2018

MacConkey’s agar with both lactose and non lactose fermenters Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Composition Uses Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Triple sugar iron A B C D E F G H Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad (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 12/1/2018

Dr. Emad AbdElhameed Morad Quiz A: B: C: D: Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Sugar media Composition Dr. Emad AbdElhameed Morad 12/1/2018

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 12/1/2018

Dr. Emad AbdElhameed Morad 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. Enrichment media Dr. Emad AbdElhameed Morad 12/1/2018

Cultivation of anaerobes Culture media for anaerobes Anaerobic jar Dr. Emad AbdElhameed Morad 12/1/2018

Culture media for anaerobes Examples of anaerobic bacteria: clostridia, bacteriodes, lactobacilli. Meat contains hematin, glutathione and lipids which reduce the oxygen in the medium. Culture media for anaerobes Robertson cooked meat medium Thioglycolate broth Deep soft agar Dr. Emad AbdElhameed Morad 12/1/2018

Robertson cooked meat medium Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad 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. Anaerobic jar Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Anaerobic jar Dr. Emad AbdElhameed Morad 12/1/2018

Dr. Emad AbdElhameed Morad Thank You Dr. Emad AbdElhameed Morad 12/1/2018