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Experiment one

Experiment one. 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) Pyogenic cocci Pathogenic enterobacteria.

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Experiment one

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  1. Experiment one • 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) • Pyogenic cocci • Pathogenic enterobacteria

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

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

  4. Streak Plate Method • PROCEDURE: • Flame your inoculating loop until the wire 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.

  5. Streak Plate Method • PROCEDURE: • Cover the petri dish, invert the plate. Sterilize the loop, label your name, date et al. • Incubate the plate at 37℃ for 18-24 hours. • Observe the bacterial colonies.

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

  7. mucoid Staphylococcus aureus Streptococcus pyogenes

  8. Agar Slope Method • MATERIALS : 1. Agar slope 2. Colonies on agar plate • PROCEDURE : 1. With the flame-sterilized wire inoculating loop, transfer a small amount of bacteria from the colony on agar plate. Then streak on the agar slope. 2. Sterile the mouth of tubes, replug the test tubes and flame the loop. 3. Label and incubate at 37℃ for 18-24 hours 4. Observe your result.

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

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

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

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

  13. Stab Culture • 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.

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

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

  16. Gram stain • 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.

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

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

  19. Use of The Oil-immersion Microscope

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

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

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

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

  24. Precautions • when lowering the tube, must look at the microscope from the side • Do not allow chemicals to contact the microscope • Clean the mechanical parts with gauze; the oil can be wiped off with lens paper. • 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.

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

  26. Laboratory Diagnosis of Pathogenic Enterobacterial Infection

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

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