Experiment 2

routine cultivation of bacteria

The cultivation of bacteria (1)

Contents1. Media for routine cultivation of bacteria2. Techniques for bacterial cultivation and inoculation3. Growth characteristics of bacteria on/in different media4. Metabolic activities of bacteria5. The physical effect on bacteria and commonly used sterilizers

1. Media for routine cultivation of bacteria Media--- The basic ingredients of media include carbon source 、 nitrogen source、 inorganic salt 、 growth factor and distilled water etc.

Classification of culture media

According to function, in general, media can be classified five types:

1. basal medium ( agar plate medium) 2. nutrient medium (blood agar plate medium) 3. selective medium 4. differential medium 5. anaerobic medium.

According to physical properties, media can be classified three types:

1. solid medium ( 1.5～ 2.5% of agar) agar plate /slant medium 2. semi-solid medium (0.3～ 0.5% of agar) 3. liquid medium

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2. Techniques for bacterial inoculation

Bacterial inoculation is a basic technique of microbiology. The operation must be under sterile condition.

1) isolation cultivation 2) pure cultivation

• 1) Isolation cultivation• streak a bacterial culture on a nutrient agar

plate to separate the individual bacterial cells, each isolated cell will develop into a pure colony after incubation.

• continuous streak method• quadrant streak dilution method

method

2) Pure cultivation transfer a single colony from agar plate or a

pure slant culture to liquid medium, slant or semisolid medium to get a pure culture.

subculture into liquid mediumsubculture into agar slant/platesubculture into agar deep tube (semi-solid)

3. Phenomena of bacterial growth

on agar plate (on blood agar plate)

in liquid medium in semi-solid medium

1) on agar plate observe the isolated colony on the surface of

nutrient agar plate; note size, shape, elevation, margin, surface, transparency and pigment (color of colony).

on agar plate -- colonycolony

Note:

Size 大小Shape 形状Elevation 突起Margin 边缘Surface 表面Transparency 透明度Pigment 色素

on blood agar plate observe hemolysis (complete and incomplete) phenomena around colony.

3) in liquid medium three growth phenomena will be seen: surface growth pellicle 表面生长 uniformly turbid 均匀混浊生长 sediment in bottom 沉淀生长

In liquid mediumsurface growth pellicle uniformly turbid sediment in bottom

4) in semi-solid medium nonmotile organism motile organism

4. Metabolic Products and Biochemical Testing

Carbohydrate Fermentation Test

溴甲酚紫【 pH 5( 黄 ) － pH7( 紫 ) 】

Methyl Red (MR) Test

Voges-Proskauer (VP) Test

Indole Test

• Citrate utilization test

I M Vi C test E.coli + + - - E.Aerogenes - - + +

Hydrogen Sulfide (H2S ) Production

sulfur amino acid

sulfur inorganic compoundH2S

Pb2+

Fe2+

PbS

FeS（ black ）

Urease Test

5. The physical effect on bacteria and commonly used sterilizers 1) Physical factor: dry 、 moist 、 ultraviolet 、 ultrasonic wave

2) Commonly used sterilizers: autoclave dry-air oven bacteriological filters

Experiment manipulation

1. Continuous streak method or quadrant streak method strain: S.aureus or E.coli one piece of plate/person

2. Observe phenomena of bacterial growth on agar plate in liquid medium in semi-solid medium

Experiment report1. Continuous streak method or quadrant streak method: principle procedure result: describe the characteristics of colony

analysis2. Draw phenomena of bacterial growth