Experiment 2

routine cultivation of bacteria

The cultivation of bacteria (1)

Contents1. Media for routine cultivation of bacteria

2. Techniques for bacterial cultivation and

inoculation

3. Growth characteristics of bacteria on/in different

media

4. Metabolic activities of bacteria

5. 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

1 2 31

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 methodmethod

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 medium

subculture into agar slant/plate

subculture 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

analysis

2. Draw phenomena of bacterial growth