lec 04. cultivation of microorganisms

2/6/2017

Cultivation of Microorganisms

Sebghatullah Mansoor

BS (Medical Sciences), MS (Microbiology),

MPH (Continue…)

Medical faculty of Malalay University

08/Feb/2017

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Fermentation

• Fermentation is a metabolic process that converts sugar to acids, gases, or alcohol.

• It occurs in yeast and bacteria

• Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium, often with the goal of producing a specific chemical product.

• French microbiologist Louis Pasteur was the discoverer of Fermentation.

— Some of the bacteria ferments a several types of sugars such as glucose, lactose, sucrose and etc…

— We can identify bacteria according to sugar fermentation.

— E. coli & Serratia both are Gram –ve rod, cause UTI but we can differentiate through sugar fermentation

— e.g., E. coli lactose fermenters and Serratia are non lactose fermenters 2/7/2017

Bacterial enzymes

• Enzymes are biological catalyst which reproduce by cells

• We can classify according to their molecular weight

– Enzymes which are made of only proteins

– Enzymes which are not made of only proteins

• Enzymes are specific and acts only on their specified substrates such as Lactase enzyme which act only on lactose that degrade to glucose and galactose .

• Enzymes are present in the cytoplasmic membrane, Ribosome, etc…

• The activity of enzymes are dependent on the culture media PH, Temperature, and the concentration of the bacterium

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• Exoenymes; which are secreted by some of the bacteria to react on the medium nutrient.

• Endoenymes; Which are produce inside the cell for metabolism of the bacterium

• Some material are added to the medium which enhance the enzymatic activity that called Adivitor. e.g., Mg, Ca

• Some material inhibit the enzymatic activity which are called inhibitors. e,g., salts and antibiotics

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Bacterial enzymes…

• Some common enzyme that produce by bacteria

― Hemolysis

― Coagulase

― Urease

― Lecithinase

― Hyaluronidase

― Collagenase

― Leucocidinase

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Bacterial enzymes…

Some of microbiology tools

• Petri dishes or plates; The light, circular, transparent dish used for culture of micro-organism

• Inoculum loop; A simple tool used to recover an inoculum from a culture of micro-organisms.

• Autoclave; A strong heated container used for chemical reactions and other process using high pressure and temperature (121 °C) (moist heat)

• Incubator; A device used to grow and maintain microbiological culture under (37 °C )

• Agar; is a jelly like substance obtained from algae, commercially available as powder and can be mixed with water for making a culture medium

• Broth; A liquid medium which composed of agar powder plus peptone+ Nacl + extract of meat and some of the proteolytic enzymes

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• Laminar flow Hood ; Laminar airflow is defined as air moving at the same speed and in the same direction, with no or minimal cross-over of airstreams (or “lamina”).

• Glass flask; Glass bottles used for sterilization of culture media

• Antibiotic discs; Commercially available small antibiotic discs• Anaerobic station; anaerobic microbiology equipment uses an oxygen-free

environment to dry nitrogen up to 99.9% pure, and delivers gas flow up to 400 SCFH• Medium; a nutrient mixture used to support microbial growth

• Sterilization methods; include autoclaving, dry-heat, filtration, UV exposure, etc…

• Culture; is the part of specimen grown in culture media.

• Culture Media: is a medium (liquid or solid) that contains nutrients to grow bacteria.

• Colony; On solid media the bacteria form pigments which are the visible form of many collocated bacteria which is called Colony2/8/2017

Some of microbiology tools

Petri dishes Inoculum loop

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Autoclave Incubator

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Incubator

Laminar flow Hood Anaerobic station

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Agar Broth

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Glass flask Antibiotic discs

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Colony

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Cultivation ; is the process of propagating organisms by providing the proper environmental conditions.

– Nutrients (Carbon, Nitrogen, Sulfur, Phosphorus, Mineral)

— PH (7.2-7.6)

— Temperature

— Aeration (Oxygen)

— Salt concentration,

— Ionic strength of the medium


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• Preparation of culture media ; Culture medium is made of broth or extracts of animal meat, some other material for bacterial growth support is added to medium.,

— Peptone

— Salt

— Serum

— Blood

Autoclave ; Moist heat about 121 °C for 15 to 30 minutes

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Different Types Of Culture Media

● According to Consistency 1. Solid medium

2. Semi Solid

3. Liquid medium

● According to Nutrition• Basic Medium

• Enriched Medium

• Selective Medium

• Indicator Medium (differential )

• Transport Medium2/7/2017

1. Solid culture media ; Media are solidified by incorporating a gelling agent such as agar or gelatin.

• Solid media are used mainly in petri dishes as plate cultures. Also in bottles or tubes as stab (deeps) or slope cultures.

2. Semi-solid culture media; This form of culture medium is prepared by adding a small amount of agar (0.4 – 0.5% w/v) to a liquid medium.

• Semi-solid media are used mainly as transport media, and for motility and biochemical tests.

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3. Liquid culture media; are most commonly used as enrichmentwhere organisms are likely to be few e.g. blood culture.

• Some organisms produce a surface growth on the medium in which they are growing e.g. Vibrio cholerae when growing in alkaline peptone water.

• Fluid media may also be used for biochemical testing

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Liquid media in tubes & Bottles Solid media in tubes

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● Basic Media ; These are simple media such as nutrient agar and nutrient

broth that will support the growth of microorganisms that do not have special

nutritional requirements.

• Some of the common bacteria can grow

– E. coli

– Staphylococcus aureus + S. albus S. citrus

– Pseudomonas

– Proteus

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● Enriched Media ; Basic media may be enriched with whole or lyzedblood, serum, peptones, yeast extract, vitamins and other growth factors

• An enriched medium increases the numbers of a pathogen by containing all the necessary ingredients to promote its growth

• Enriched media are required for the growth of fastidious organisms as H. influenzae, Neisseria species, and some Streptococcus species.

• e.g,. Blood agar, chocolate agar

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• Blood agar; commercially available nutrient ager powder is added to glass flask and distilled water then autoclave at 121 °C for 20 minutes after that take out the glass flask, wait till the temperature down to 25 °C then add 5-10% of sheep blood, If the blood is added at 50 °C then the blood cells will lyse which is called Chocolate agar;

• Some bacteria have hemolytic enzymes that hemolyse the RBCs in Blood agar which are classify in three types

1. Alpha hemolysis: which partially hemolysis occurs. e.g., S. pneumonia,

2. Beta hemolysis: which clear hemolysis occurs. e.g., S. pyogenes

3. Gama hemolysis: which no hemolysis occurs. e.g., S. aureus 2/7/2017


● Selective Media; These are solid media which contain substances (e.g. bile salts or other chemicals, dyes, antibiotics) which inhibit the growth of one organism to allow the growth of another to be more clearly confirmed.

• A selective medium is used when culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants overgrowing a pathogen.

• e.g. V. cholerae can be isolated on an alkaline medium such as TCBS agar.

• Temperature may also help to select an organism e.g. Listeria monocytogenes can grow at 4 °C whereas other organisms are inhibited.

• TSI (triple sugar iron agar): slanted tube.

• EMB (Eosin Methylene blue): enteric isolation media. 2/7/2017


• TSI (Triple sugar iron):

• This media is composed of, Lactose, Sacros, Glucose

Phenol, Ferrous, Proteins and Sulfate.

― Differentiate bacteria according to sugar fermentation

― e.g,. Differentiate Salmonella or Shigella from other enteric flora

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TSI tubes

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● Indicator (differential) media; These are media to which dyes or other substances are added to differentiate micro-organisms.

• Many differential media differentiate between bacteria by adding an indicator which changes colour when acid is produced following fermentation of a specific carbohydrate e.g. MacConkey agar.

• Note: Many media used to isolate pathogens are both selective and enrichment or both selective and differential.

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• MacConkey’s agar;

• This media is composed of Nutrient agar, Bile salt, Lactose, Water, Nacl

• Properties

― Differentiate bacteria according to their sugar fermentation,.

• Lactose fermenters bacteria . e.g,. Klebsiella, E. coli, etc

• Lactose non fermenters bacteria . e.g,. Shigella, Serratia, etc

• Late lactose fermenters bacteria e.g,. V. cholera, Salmonella sonnei, etc

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Lactose fermenters Non- Lactose fermenters

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● Transport media; These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection.

• Their use is particularly important when transporting microbiological specimens from health centers to the district microbiology laboratory or specimens to the Regional Public Health Laboratory.

• Examples;

— Cary-Blair medium for preserving enteric pathogens

— Amies transport medium for ensuring the viability of gonococci.

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THANK YOUQuestions ?

2/6/2017

lec 04. cultivation of microorganisms

Health & Medicine