16
1 Cultivation of microorganisms

Cultivation of Microorganisms

Embed Size (px)

Citation preview

Page 1: Cultivation of Microorganisms

1

Cultivation of microorganisms

Page 2: Cultivation of Microorganisms

2

Media - the nutrient preparations that are used for culturing microorganisms

Properties of Media:1.should be nutritive (contains the required amount of nutrients)- proteins, fats, carbohydrates, or lipids, etc.2.contain mineral salts – sulphate, phosphates, chlorides, carbonates of K, Ca, Mg3.a proper moisture4.a suitable pH – 7,2-7,45.accesory growth factors – tryptophan for Salmonella typhi or X & V factors for Haemophilus influentzae6.sterile (initially)

Page 3: Cultivation of Microorganisms

3

Culture Media Consistency Classification

•solid media - provide a firm surface on which cells can form discrete colonies - usually used as: slants, stabs, petri dishes (ex.: Coagulated blood serum)

Three physical forms are used:

•liquid, or broth, media- − water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted (ex.: Nutrient broth, Peptone solution, etc.);

•semisolid media - used to determine the motility of bacteria and to localize a reaction at a specific site (ex.:; Cystine trypticase agar medium)

Culture Media Complexity Classification

1.Simple media – consists of only basic necessitie; used to culture non-fastidious bacteria (agar plate; nutrient broth; peptone water)2.Complex media - for nutritionally more exigent bacteria

Page 4: Cultivation of Microorganisms

4

Bacteriological Media Application Classification

1. Basic media – for the general cultivation of bacteria (agar plate, bouillon, peptone water)

2. Storage media - medium in which bacteria are stored in "stock culture" (Yeast extract mannitol agar medium)

3. Enrichment media – used to select a desired organism from a specimen with mixed flora (addition of extracts of plant or animal tissues to Nutrient broth or Nutrient agar media provides additional nutrients and the media start favouring the growth of fastidious heterotrophic bacteria)

Nutrient broth

Page 5: Cultivation of Microorganisms

5

4. Selective and differential media

• Selective media – are growth media that contains substances that inhibit the growth of unwanted organisms but permit the growth of the desired organism; uses certain dyes, high salt concentration, pH or antibiotics

• Differential media - supports the growth of several types of microorganisms, but it is designed to highlight differences among these microorganisms; contains a combination of nutrient and pH indicators to visually differentiate bacteria that grow on or in it

• Examples: Mannitol salts agar (selects against non-skin flora; for the selection of the

S.aureus; mannitol fermentation = yellow)

Page 6: Cultivation of Microorganisms

6

MacConkey agar (selects against gram-positives; for the selection of the Enterobacteriaceae and related gram-negative bacilli;) (lactose fermentation = red)

•Salmonella-Shigella medium (a high selective medium; inhibit the growth of most coliform organisms; permit the growth of Salmonella and Shigella; lactose fermentation = red)

Page 7: Cultivation of Microorganisms

7

Bile Esculin Agar (used to identify members of the genus Enterococcus (E faecalis and E. Faecium); tests the ability of organisms to hydrolyze esculin in the presence of bile = brown to black)

Biochemical Test Media – used to isolate and identify bacteria from clinical specimens; contain a sugar ( glucose, lactose, sucrose, etc.) or another biochemical substance such as: indole, methyl red, citrate (Simmon’s Citrate Agar), urea, and a colour indicator. Ex.: TSI (triple sugar iron), SIM (Sulfur Indole Motility),etc.

TSI SIM

urea

se t

est

Page 8: Cultivation of Microorganisms

8

Anaerobic media – are nutritional base media used for the cultivation of anaerobic bacteria (will grow only in the absence of O2) from clinical specimens ( liqiuds or solids- schaedler blood agar)

Media for antibiotic susceptibility testing - Mueller-Hinton agar

Transport medium – is a special purpose media that contains balanced salts to protect the specimen from pH changes and keeps the swab moist while in transit to the laboratory for culturing ( ex. Cary- Blair transport medium for the enteric pathogens).

schaedler blood agar

BHI

Cary- Blair→

Amies→

Stuart→

Page 9: Cultivation of Microorganisms

9

Isolation of bacteria in pure culture Common Methods of isolation of pure culture Liquid media

Page 10: Cultivation of Microorganisms

10

Solid media

• Streak plate technique • Inoculation -on the surface of a solid medium in a Petri dish• Rational of procedure - to physically separate the various cells one from another by spreading

them over solid surface• The bacterial suspension is streaked on an agar plate with the aid of an inoculating loop. • As the streaking goes on, fewer and fewer cells are left on the loop and finally single cells

attach to the surface of the medium.

Page 11: Cultivation of Microorganisms

11

Inoculating a deep

Sterilize the needle (until red hot), wait a few seconds and than pick your sample, stab the needle in the middle of the deep and remove it through the same stab.

Inoculating an agar slant

Sterilize the loop (until red hot) and wait a few seconds. Touch the loop to an isolated colony and carefully place it into the sterile slant tube. Using a zig-zag motion, move the inoculating loop along the SURFACE of the sterile agar slant from the bottom of the slant up to the top so that a maximum surface area is exposed to the bacterial culture.

Page 12: Cultivation of Microorganisms

12

Isolating anaerobic bacteria 

• Oxygen within the jar and the hydrogen that is generated are converted to water in the presence of the catalyst, thus producing anaerobic conditions.

• Carbon dioxide - required for growth by some anaerobes and stimulates the growth of others.

2. evacuation and replacement

• air is evacuated from the sealed jar containing the culture plates and is replaced with an oxygen-free mixture of 80 percent nitrogen, 10 percent hydrogen, and 10 percent carbon dioxide.

Solid media

•the anaerobic jar - It is a medium-sized glass or plastic jar with a tightly fitting lid.It can be set up by two methods:

1.a commercially available hydrogen and carbon dioxide generator envelope (GasPak) is placed in the jar along with the culture plates the generator is activated with water

anaerobic jar

Page 13: Cultivation of Microorganisms

13

• Liquid media

• Heat a broth tube to boiling to drive off all oxygen (10-20 min)

• Cool it to 45 to 50o C in water;

• Add the sample that may contain anaerobe;

• Use your loop- stir a bit to disperse the bacteria without getting air back into the tube.

• 2 ml of paraffin oil is added to seal the medium from the air

• Incubate ;Aspects of bacterial growth

Liquid media

• Growth of bacteria is shown by turbidity in medium.

Page 14: Cultivation of Microorganisms

14

Solid mediaBacteria  grow on solid media as colonies. A colony - a visible mass of microorganisms all originating from a single mother cellBasic elements - for all colonies:•Surface – smooth (S), glistening (G), rough (R), mucoid (M), etc.•Form – the basic shape of the colony- circular, filamentous,etc.•Elevation - the cross sectional shape of the colony•Margin - the magnified shape of the edge of the colony•Opacity - transparent (clear), opaque, translucent , etc•Pigmentation – white ( S.epidermidis), buff, red, purple, etc.•Consistency•Odor •Mobility

Page 15: Cultivation of Microorganisms

15

Cultivation of viruses

• can replicate only in receptive living cells

• these are provided by cell cultures, embrionated eggs or animals.

• Cell cultures: in vitro biological system formed by isolated viable cells that maintain their capability to multiply;

• to retain the cells viability nutrients, energy source, aseptic conditions, proper temperature, pH must be provided.

primary cultures – derived from freshly removed animal/human, normal/tumor tissue, obtained by dispersing cells; cells in primary cultures are unable to grow for more than a few passages;

diploid cell cultures (secondary cell cultures) – derived from embryos – cells are viable for about 50-60 passages

continuous cell lines – derived from malignant tissues, capable for indefinite growth o ex. HeLa, Vero, Hep2, MacCoy, etc.

Page 16: Cultivation of Microorganisms

16

Detection of virus-infected cells:

- cytopathic effect: cell lysis, necrosis, inclusion formation, giant cell formation,cytoplasmic vacuolization- adsorbtion of eryhrocytes to infected cells, called hemadsorbtion, due to the expression of a viral protein (hemagglutinin – influenza virus) on the membrane of infected cells; this becomes positive before cytopathic effects become visible- detection of virus-specific nucleic acid