Experiment one Basic methods of microbiological laboratory Basic methods of microbiological laboratory  Bacterial culture  Staining of bacteria  Use of oil-immersion microscope  Observation of several special bacteria Isolation and culture of bacteria(streak plate culture) Isolation and culture of bacteria(streak plate culture)  Pyogenic cocci  Pathogenic enterobacteria

Bacterial Culture and Growth State Observation PURPOSE : PURPOSE : To obtain information about bacterial biological characteristics To obtain information about bacterial biological characteristics METHODS : METHODS : streak plate method isolation and culture streak plate method isolation and culture agar slope method agar slope method liquid medium culture pure culture liquid medium culture pure culture stab culture stab culture anaerobic culture anaerobic culture

Streak Plate Method PURPOSE: PURPOSE: Isolation and culture of bacteria growing together in a specimen Isolation and culture of bacteria growing together in a specimen MATERIALS: MATERIALS: l. Mixed broth culture of Escherichia coli l. Mixed broth culture of Escherichia coli and staphylococcus aureus. and staphylococcus aureus. 2. Nutrient agar plate. 2. Nutrient agar plate.

Streak Plate Method PROCEDURE: PROCEDURE:  Flame your inoculating loop until the wire glows red. glows red.  Allow the loop to cool and get a loopful of the suspension of sample.  Pick up your plate and streak the surface, Flame the loop before streaking next section. When streaking, be care not to cut into the agar and not to be far away from flame.

Streak Plate Method PROCEDURE: PROCEDURE:  Cover the petri dish, invert the plate. Sterilize the loop, label your name, date et al.  Incubate the plate at 37 ℃ for hours.  Observe the bacterial colonies. colonies.

Streak Plate Method RESULT: RESULT: observe the location and characteristics of the bacterial colonies: observe the location and characteristics of the bacterial colonies: Size Size Shape: circular, irregular, spreading Shape: circular, irregular, spreading Color Color Density: transparent, or opaque Density: transparent, or opaque Elevation Elevation Margin Margin Hemolysis Hemolysis Surface: rough or smooth, dry or moist. Surface: rough or smooth, dry or moist. Bacillus subtilis Proteus vulgaris

Streptococcus pyogenes Staphylococcus aureus mucoid

Agar Slope Method MATERIALS : MATERIALS : 1. Agar slope 1. Agar slope 2. Colonies on agar plate 2. Colonies on agar plate PROCEDURE : PROCEDURE : 1. With the flame-sterilized wire inoculating loop, 1. With the flame-sterilized wire inoculating loop, transfer a small amount of bacteria from the transfer a small amount of bacteria from the colony on agar plate. Then streak on the agar slope. colony on agar plate. Then streak on the agar slope. 2. Sterile the mouth of tubes, replug the test tubes and 2. Sterile the mouth of tubes, replug the test tubes and flame the loop. flame the loop. 3. Label and incubate at 37 ℃ for hours 3. Label and incubate at 37 ℃ for hours 4. Observe your result. 4. Observe your result.

Agar Slope Method RESULTS : RESULTS : There are many similar wet colonies on the surface. If there are some other forms, it indicates culture sample is not pure. There are many similar wet colonies on the surface. If there are some other forms, it indicates culture sample is not pure.

Liquid Medium Culture MATERIALS ： MATERIALS ： 1. Peptone water 1. Peptone water 2. Colonies on agar plate 2. Colonies on agar plate PROCEDURE ： PROCEDURE ： 1. Flame -sterilize the wire inoculating loop. 1. Flame -sterilize the wire inoculating loop. 2. Insert the wire loop containing a small amount 2. Insert the wire loop containing a small amount of bacteria into the liquid culture robe. of bacteria into the liquid culture robe. 3. Scratch the wall of tube over the broth in order 3. Scratch the wall of tube over the broth in order to let bacteria drop into the liquid. to let bacteria drop into the liquid.

Liquid Medium Culture PROCEDURE ： PROCEDURE ： 4. Flame the mouth of the tube and reinsert the 4. Flame the mouth of the tube and reinsert the cotton plug. Flame-sterilize the wire loop. cotton plug. Flame-sterilize the wire loop. 5. Label the tube, incubate at 37 ℃ for 24 hours 5. Label the tube, incubate at 37 ℃ for 24 hours 6. Observe the result. 6. Observe the result. RESULTS: RESULTS: turbid, flocculent, pellicle turbid, flocculent, pellicle

Stab Culture METHODS: METHODS: 1. Flame-sterilize inoculating needle. 1. Flame-sterilize inoculating needle. 2. Insert the needle with a small bacteria to the 2. Insert the needle with a small bacteria to the center of the culture, be care not to touch the center of the culture, be care not to touch the bottom of the tube, then draw it out in the same way. bottom of the tube, then draw it out in the same way. 3. Flame the mouth of the tube and reinsert the cotton 3. Flame the mouth of the tube and reinsert the cotton plug. Flame-sterilize the needle. plug. Flame-sterilize the needle. 4. Label the tube, incubate for 24 hours at 37 ℃. 4. Label the tube, incubate for 24 hours at 37 ℃. 5. Observe the result. 5. Observe the result.

Stab Culture RESULTS: RESULTS: Motile bacteria will migrate from the line of inoculation to form a diffuse turbidity in the surrounding medium; nonmobile bacteria will grow only along the line of inoculation. Motile bacteria will migrate from the line of inoculation to form a diffuse turbidity in the surrounding medium; nonmobile bacteria will grow only along the line of inoculation.

Staining of Bacteria PURPOSE : PURPOSE : To make bacteria more easily observable To make bacteria more easily observable To acquaint you with Gram stain To acquaint you with Gram stain MATERIALS: MATERIALS:  Simple stain  Gram stain  Acid-fast stain  Special stain  Spore stain  Capsule stain  Flagella stain  Metachromatic granules stain.

Gram stain purpose: purpose: differentiating bacteria differentiating bacteria MATERIALS : MATERIALS :  Slant cultures of and Escherichia coli and S.aureus (18 to 24 hours old)  Crystal violet, iodine solution, 95% alcohol, safranin  Microscope slides

Gram stain PROCEDURE: PROCEDURE:  Smear: size of a dime to form a thin film  Dry : air dry  Fix: through the warm air above the flame two or three times.

Gram stain PROCEDURE: PROCEDURE:  Stain  primary stain : crystal violet ， 60 seconds, Wash with water Wash with water  mordant stain : iodine solution, 60 seconds, Wash with water Wash with water  decolorize : 95% alcohol, 20 seconds Wash with water Wash with water  Counterstain : safranin 30 seconds Wash with water Wash with water  Dry and examine: oil immersion lens

Gram stain results: results:  typical shapes and colors of the bacteria.  Gram positive bacteria are violet colored and Gram negative cells are red colored.

Use of The Oil-immersion Microscope

study morphology study morphology structure structure staining characteristics staining characteristics motility of different microorganisms. motility of different microorganisms. Use of The Oil-immersion Microscope Microscope

MATERIALS Microscope, immersion oil, lens Microscope, immersion oil, lens paper, glass slides, and cover slips. paper, glass slides, and cover slips. Prepared stained slides of several Prepared stained slides of several types of bacteria. types of bacteria.

Types of objective lense 1. the low-power objective lense 1. the low-power objective lense the 10× or 16-millimetre(mm) the 10× or 16-millimetre(mm) 2. the high-dry one 2. the high-dry one the 40×,50×,or 4mm objective the 40×,50×,or 4mm objective 3. the oil immersion 3. the oil immersion the 90×, 100×, or 1.8 mm objective. the 90×, 100×, or 1.8 mm objective.

PROCEDURE Carefully place the microscope on the desk and Carefully place the microscope on the desk and examine it examine it preparation: select the objective lense preparation: select the objective lense Make the iris diaphragm open Make the iris diaphragm open and the top of the condenser at and the top of the condenser at the level of the stage. the level of the stage. place a drop of immersion oil on the cover glass place a drop of immersion oil on the cover glass of the microslide of the microslide lower the oil immersion objective tube while until lower the oil immersion objective tube while until it engages the drop it engages the drop

Precautions when lowering the tube, must look at the microscope when lowering the tube, must look at the microscope from the side from the side Do not allow chemicals to contact the microscope Do not allow chemicals to contact the microscope Clean the mechanical parts with gauze; the oil can be Clean the mechanical parts with gauze; the oil can be wiped off with lens paper. wiped off with lens paper. To remove immersion oil from the optical glass parts, To remove immersion oil from the optical glass parts, wipe with lens paper moistened with xylol. Discard the lens paper. Wipe rapidly to prevent injury to optical glass settings. wipe with lens paper moistened with xylol. Discard the lens paper. Wipe rapidly to prevent injury to optical glass settings.

focus upward slowly until the image appear. focus upward slowly until the image appear. Complete focus with the fine adjustment Complete focus with the fine adjustment Clean the microscope when this experiment is Clean the microscope when this experiment is ended ended  Leave the microscope with the lowest power objective in the working position objective in the working position  Remove immersion oil from prepared practice slides. Return these to instructor. slides. Return these to instructor.  Place a plastic cover over the microscope or place it in its case. Record the status of the place it in its case. Record the status of the microscope. microscope.

Laboratory Diagnosis of Pathogenic Enterobacterial Infection

Colonial characteristic observation Specimens isolation Gram Staining (SS/EMB plate) Serological identification TSI Biochemical reaction