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Contents
Exp.1． Bacterial Morphology ………………………………………(1)
Exp.2.
Bacterial Physiology ………………………………………(4)
Exp.3.
Bacteria Distribution ………………………………………(8)
Exp.4.
Enumeration of Microorganisms ………………………… (9)
Exp.5.
Physical Effects on Bacteria………………………… (11)
Exp.6. Effects of chemical disinfectant on Bacteria…………(14)
Exp.7.
Microbial Antibiotic Susceptibility Testing………… (15)
Exp.8.
Bacteriophages ………………………………………………(18)
Exp.9.
Bacterial Variation……………………………………… (20)
Exp.10. Bacterial Pathogenicity………………………………… (22)
Exp.11. Bacterial Plasmid DNA Extraction………………………… (23)
Exp.12. Pathogenic Cocci…………………………………………… (25)
Exp.13. Enteric Bacilli………………………………………………… (27)
Exp.14. Clostridium……………………………………………………(30)
Exp.15. Mycobacterium…………………………………………………(31)
Exp.16. Polymerase Chain Reaction ( PCR ) ………………… (32)
Exp.17. Mycoplasma…………………………………………………… (35)
Exp.18. Rickettsiae……………………………………………………(35)
Exp.19. Chlamydia……………………………………………………… (36)
Exp.20. Spirochetes……………………………………………………(36)
Exp.21. Actinomycetes……………………………………………… (36)
Exp.22. Fungi…………………………………………………………… (37)
Exp.23. Viral Morphology…………………………………………… (39)
Exp.24. Culture of Aminal Virus………………………………… (39)
Exp.25. Viral Serology……………………………………………… (42)
Exp. 1．Bacterial Morphology
Content
1. Use oil-immersion lens of microscope
2. Gram stain
3. Observation of bacterial shape and special structure
4. Observation of the living bacteria
Use oil-immersion lens of microscope
[Principle]
The body of bacterium is tiny. It must be observed under the
oil-immersion lens of microscope that can magnify the object about
1000 diameters. A drop of cedar oil is placed between specimen and
oil lens when oil- immersion lens is used. Because the index of
refraction of cedar oil (n=1.515) approximates to that of slide
(n=1.52), the refraction caused by different densities of media
(e.g., nair=1.0) can be weakened as far as possible which can avoid
light dispersion and make the field of vision brighter and object
more clear. Refer to the Fig 1.
Fig 1. principle of oil-immersion lens
2
[Material]
(1) specimen
(2) microscope、cedar oil、xylene、lens paper
[Procedure]
(1) Once the specimen has been brought into sharp focus with a low
powered lens, preparation may be made for visualizing the
specimen under oil-immersion. A drop of oil is placed on the
slide directly over the area to be viewed. The nosepiece is
rotated until the oil-immersion objective locks into position. The
slide is observed from the side as the objective is rotated slowly
into position. This will ensure that the objective will be properly
immersed in the cedar oil. The fine adjustment knob is
readjusted to bring the image into sharp focus.
(2) On completion of the laboratory exercise, the following steps
are recommended:
a. All lens are cleaned with dry, clean lens paper. Xylene is
used to remove dried cedar oil from the lens;
b. The low power objective is placed in position and the body
tube is lowered completely;
c. The microscope is returned to the cabinet in its original
condition.
Gram Stain
[Principle]
It is necessary to stain bacteria before they can be seen with
light microscope. One of the most important stain methods used
is the Gram Stain which differentiates bacteria into two broad
groups. Gram positive organisms appear dark purple, while
Gram negative organism appear pink.
3
[Material]
(1) slant agar culture of s.aureus and proteus
(2) Gram stain reagents: crystal violet, iodine solution, 95%
alcohol, diluted carbon fuchsin
(3) NS
[Procedure]
1. Smear preparation:
(1) add a loop of NS on a slide
(2) remove the cotton (or plastics) plug from the mouth of the
culture; tube, take a small inoculum from slant agar with a
sterile loop, return the cotton plug, then emulsify the
inoculum in the drop of NS, spread the drop out thinly until
almost dry, flame the loop;
(3) dry the smear in the air or high above the alcohol burner;
(4) fix the smear by passing the slide smear side up, quickly
through the flame two or three times;
(5) outline the area of the smear with a glass marking pencil on
the slide.
2.Stain procedure:
(1) add crystal violet, stain for 1 min, then rinse the slide by
washing the stain off with a jet of tap water;
(2) add iodine solution, let the slide stand for 1 min, rinse with
water;
(3) decolorize the stain by letting 95% alcohol run down over the
slide, until the stain no longer is being removed from the slide.
It takes about 30 secs, quickly rinse the slide with water;
(4) add diluted carbol fuchsin and stain for 30 secs, rinse the slide
with water;
(5) dry the slide with blotting paper. Examine the smear under oil
immersion lens.
4
Observation of bacterial shapes and special structures
Hanging-drop preparation
[Material]:
1. Broth culture of Staphylococcus aureus and Pseudomonas
aeruginosa, cultured 6-8 hrs.
2. Hollow ground slide, coverglass , petrolatum
[Procedure]:
1. A drop of liquid culture in placed in the center of a coverglass.
2. A special slide with a hollow depression in the center is ringed
with a thin layer of petrolatum , and then turned upside down
over the coverglass, so that the drop of bacteria is in the center
of the depression.
3. The entire slide with the coverglass then is quickly turned over,
so that, the drop of bacteria actually hangs from the coverglass
down into the depression.
4. The slide is placed on the stage of a microscope and bacteria are
observed using high dry objective.
Note : Keep in mind that a bacterium is considered motile only
if it seems to be going in a definite direction . Even nonmotile
bacteria will bounce back and forth due to bombardment from
molecules of water (Browniam movement)
Pressing –drop preparation
[Material ]:
1. Broth culture of S.aureus and P.aeruginosa
2. Slide, coverglass
[Procedure] :
1. place several drops of bacterial culture on the common slide.
2. put a coverglass on the culture .
3. Examine under microscope.
(Cheng Yizhe)
5
Exp. 2. Bacterial Physiology
Content:
1.Media for routine cultivation of bacteria
2.Techniques for bacterial inoculation
3.Growth characteristics of bacteria on/in different media
4.Metabolic activities of bacteria
Media for routine cultivation of bacteria
The basic ingredients of media include carbon source 、
nitrogen source、inorganic salt、growth factor and distilled water.
According to function, in general, media can be classified five types:
basal medium, nutrient medium, differential, selective medium and
anaerobic medium. According to physical properties, media can be
classified three types: solid medium, semi-solid medium and liquid
medium.
[Material]
(1) beef extract
(2) peptone
(3) sodium chloride
(4) distilled water
(6) agar
0.3g
1.0g
0.5g
100ml
[Procedure]
1. liquid medium:
Dissolve the ingredients: beef extract, peptone and sodium
chloride in distilled water, adjust to pH 7.6 with 1 mol/l NaOH,
dispense in test tubes, sterilize by autoclaving.
6
2. semi-solid medium
The agar in the medium is 0.3~0.5%, the medium is dispensed
into test tubes, the other procedures are the same as those of the
liquid medium.
Note: agar, a polysaccharide extract of marine algae, is an
excipient. It is uniquely suitable for microbial cultivation, because
it melts at 100℃, but does not gel until cooled below 45℃.
3. solid medium:
To make agar plate or slant , add 2~3% agar to the medium, the
other procedures are almost the same as those of the liquid
medium. For the agar, it is poured into a sterile petri dish, after
cooling the agar will solidify to for a plate. To make agar slant, it
is dispensed into test tubes, after autoclaving the tubes are laid at
an angle so that slants ate formed after cooling of the agar.
Techniques for bacterial inoculation
Bacterial inoculation is a basic technique of microbiology.
The operation must be under sterile condition.
Ⅰ. aerobic 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.
[Material]
(1)slant culture of staphylococcus aureus or proteus
(2)agar plate
[Procedure]
(1) continuous streak method
7
remove the cotton plug or the cap from the mouth of the slant tube,
take the culture of the bacteria;
a loop of inoculum of the culture is placed on the surface of the
agar plate, near but not touching the edge, with the loop flat against
the agar surface, the inoculum is streaked on the plate surface by
moving the loop, the inoculum is back and forth continuously until the
whole plate is inoculated with bacteria. (fig. 2)
(1) Start of streak
(2) Dense area of growth
(3) Less dense area of growth
(4)Single, isolated colonies
Fig.2 continuous streak method
(2) quadrant streak dilution method
a loop of inoculum of the culture is placed on the surface of the
agar plate. the inoculum is streaked in the four quadrants of the
plate with the loop sterilized for each streak. (fig. 3)
a
b
d
c
Fig. 3 quadrant streak dilution method
8
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.
[Material]
(1) slant culture of staphylococcus aureus or proteus
(2) agar slant, liquid, semi-solid and solid medium
[Procedure]
(1) subculture into liquid medium
transfer a small number of living bacteria to the liquid.
Immerse your loop with the inoculum in the liquid and shake it
slightly to dislodge the organisms into the liquid.
(2) subculture into agar slant
a loop of inoculum is drawn lightly over the surface upside in
a straight or zigzag line.
(3) subculture into agar deep tube(semi-solid)
take the culture with a sterile inoculating needle, stab the
center of the medium vertically and insert the needle to the
bottom of the tube in a straight line, and rapidly withdraw it
along the line of insertion.
Ⅱ. anaerobic cultivation
1.biological method:
beef medium: beef as a reducing agent absorb the free oxygen,
forming an anaerobic enviroment.
2.physical method
anaerobic jar: the O2 in the jar is removed with a high vaculum
pump, then the jar is refilled with N2 or O2 and sealed.
3.chemical method------Metagallic acid methed
Streak anaerobes on a blood agar plate, invert the dish;
Put a little powder of pyrogallic acid on a thin layer of cotton
9
or gauze which has been stick to a square of glass;
Add several drops of 10% NaOH or KOH, immediately remove
the cover of the plate and place the dish on the square of glass,
seal it tightly with wax, then incubate , The chemicals absorb free
O2, producing anaerobic environment.
Growth characteristics of bacteria on/in different media
1.on agar plate
observe the isolated colonies on the surface of a nutrient agar
plate, note their size, shape, elevation, margin, surface, transparency
and pigment (color of colony).
2.on blood agar plate
observation of hemolysis(complete and incomplete) phenomena
around colonies.
3.in liquid medium (fig.4)
three growth phenomena will be seen:
(1) turbid—growth well dispersed
(2) sediment—growth at bottom
(3)pellicle—growth at surface
Fig.4
Bactarial growth in borth
10
4.in semi-solid medium (fig.5)
nonmotile organisms grow only along the line of inoculation, the
medium will remain clear away from the stab line, while the
growth of motile organisms runs out from the line of inoculation
they may even grow through out the medium. The growth pattern
gives the appearance of a brash, the medium becomes turbid.
Nonmotile growth
Motile growth
Fig.5 Bacterial growth in semi-solid medium
(Cheng Yizhe)
11
Exp.3. Bacteria Distribution
Content
1.bacterial culture from air
2.bacterial culture from finger skin
Bacterial Culture from Air
[Material]
agar plate medium
[Procedure]
(1) Open two or three agar plates, expose them to the air at the
different places for 5-10 mins, then cover them. there is a
covered agar plate as the control experiment.
(2) Incubate them at 37℃, for 18-24 hrs, observe the result.
Bacterial Culture from Finger Skin
[Principle]
Comparing the number of bacterial colonies before disinfecting
finger with those after, you will obtain such conclusion: bacteria
exist normally on the skin and the disinfectants have the effect of
killing and inhibiting bacteria.
[Material]
(1) agar plate
(2) 75% alcohol,2%iodine tincture
(3) sterile cotton swab
[Procedure]
(1) Mark the plate into four sectors with a glass pencil.
(2) Student press slightly on two sectors with forefinger, then
disinfect the finger with iodine tincture and alcohol, press on
the other sectors.
(3) Incubate agar plate at 37℃, for 18-24 hrs, observe the result.
(Cheng Yizhe)
12
Exp.4.
Enumeration of Microorganisms
Enumeration of microorganisms is especially important in dairy
microbiology, food microbiology, and water microbiology.
The Plate Count ( Viable Count )
The number of bacteria in a given sample is usually too great to
be counted directly. However, if the sample is serially diluted and
then plated out on an agar surface in such a manner that single isolated
bacteria form visible isolated colonies (see Fig.6) the number of
colonies can be used as a measure of the number of viable (living)
cells in that known dilution. We generally determine the number
of Colony-Forming Units (CFUs) in that known dilution. By
extrapolation, this number can in turn be used to calculate the
number of CFUs in the original sample.
[Materials and Organism]
1. 6 tubes each containing 0.9 ml sterile saline
2. 3 plates of nutrient agar
3. 2 sterile 1.0 ml pipettes ,Tip heads.
4. 1.5 ml EP Tube
5. bent glass rod
6. dish of alcohol
7. broth culture of Escherichia coli
13
[Procedure] (to be done in pairs)
1. Take 6 dilution tubes, each containing 0.9 ml of sterile saline.
Aseptically dilute 0.1 ml of a sample of E. coli as shown in Fig 7.
2. Using a new 1.0 ml pipette, transfer 0.1 ml from each of two
dilution tubes onto the surface of the corresponding plates of
nutrient agar.
Fig. 6: Bacteria form isolated colonies procedure
Fig. 7: Plate count dilution.
3. Using a turntable and sterile bent glass rod , immediately spread
the solution over the surface of the plates as follows:
4. Incubate the 3 agar plates upside down at 37°C until the next lab
period.
[Results]
1. Choose a plate that appears to have between 30 and 300 colonies.
2. Count the exact number of colonies on that plate using the colony counter.
3. Calculate the number of CFUs per ml of original sample as follows:
number of CFUs per ml of sample (CFUs/ml) = number of colonies in
plate ≅10≅ the dilution factor of the plate.
14
[Notes]
1. For a more accurate count it is advisable to plate each dilution
induplicate or triplicate and then find an average count.
2. Sterilize the glass rod by dipping the bent portion in a dish of
alcohol and igniting the alcohol with the flame from your burner.
Let the flame burn out.
3. Normally, the bacterial sample is diluted by factors of 10 and
plated on agar. After incubation, the number of colonies on a
dilution plate showing between 30 and 300 colonies is
determined. A plate having 30～300 colonies is chosen because
this range is considered statistically significant. If there are less
than 30 colonies on the plate, small errors in dilution technique
or the presence of a few contaminants will have a drastic effect
on the final count. Likewise, if there are more than 300 colonies
on the plate, there will be poor isolation and colonies will have
grown together.
4. Since only 0.1 ml of the bacterial dilution is placed on the plate,
the bacterial dilution on the plate is 1/10 the dilution of the tube
from which it came.
[Discussion]
1. State the formula for determining the number of CFUs per ml of
sample when using the plate count technique.
2. State the principle behind the plate count method of enumeration.
(Luan Yi)
15
Exp.5.
Physical Effects on Bacteria
Content
1.commonly used sterilizers
2.bacteriological filters
3.effects of ultraviolet radiation
Commonly Used Sterilizers
1. Autoclave:
The autoclave is essentially a large pressure cooker. When the
autoclave is closed and the stream is turned on , it becomes a sealed
container. As the steam enters, pressure builds up in the sealed
autoclave. As the pressure is increases above normal atmospheric
pressure, the temperature is raised. Normally, all forms of life are
killed by maintaining a temperature of 121.3℃ (102.97Kpa or 1.05kg/cm2)
for 15~30 mins. This cycle is often expresses as 15 lbs/in2 for 15 mins.
Culture media, solutions and cotton ( in fact, most objects
sterilized in the microbiology lab except glassware
easily
decomposed by heat) are sterilize surgical packs, towels and patient
drapes in the autoclave. Some media such as certain fermentation
broths deteriorate at 121℃, they must be autoclaves at lower
temperature, therefore at lower pressure.
The following table shows pressures and temperatures that
should help you to determine the pressure that you must maintain at
your altitude in order to achieve the necessary temperature for sterilization.
16
Table 1. autoclave steam pressure and corresponding temperature
Steam pressure
temperature℃
2
lbs/in
kg/cm2
5
0.3
108.4
8
0.6
112.6
10
0.7
115.2
15
1.1
121.3
20
1.5
126.0
25
1.8
130.4
30
2.2
134.5
2.Arnold steamer:
It is used for intermittent sterilization(factional moist heat
sterilization). In arnold steamer, like common steamer the
temperature reaches the top of 100℃, kills bacterial vegetative
bodies, but not endospores.
Place the materials to be steriliged in the steamer, turn on the
electric switch, maintain a temperature of 100℃ for 30 mins, then
take materials out, incubate at 37℃ overnight, in order to let the
endospores sprout to vegetative bodies. Repeat the procedure for 3
times, sterilization will be complete.
The method is suitable for sterilization of certain media which
contain components easily destroyed by high temperature during
autoclaving.
3. dry-air oven:
it is used for glassware, petrolatum etc. dry sterilization is
preferred for glassware because there is no condensation of
moisture. High temperature must be maintained for long periods of
time to achieve sterilization with dry heat. The routine sterilization
17
of glassware in the dry—air oven requires a temperature of
160~180℃ for 2 hrs. in addition, it is used to destroy endotoxin (or
pyrogen).
Bacteriological Filters
They are made of porcelain, asbestos and sintered glass. Inside
the filter, funnel is a membrane made with tiny pores of exactly
specified and controlled size, these pores are usually 0.45μm. the
average bacterial cell is larger than this so they cannot pass through
the filter membrane.
The filter funnel assembly and filter flask are wrapped and
autoclaved before use. When they are setup, the solution to be
sterilized is poured into the filter, a vacuum pump is attached to the
side arm of the flack. As the flask is evacuated, the solution is
drawn rapidly through the filter membrane. Bacteria are retained in
the funnel.
All the bacteriological filters operate on the same principle
regardless of the substance used for the filter. The seitz filter, which
employs on asbestos pas, is in common use. Other filters made of
sintered glass or porcelain are made in form of candles instead of a
membrane or pad.
Effects of Ultraviolet Radiation
[Material]
1.18~24hrs slant cultures of staphylococcus aureus
2.nutrient agar plate
3. 15watt low pressure mercury vapor lamp
4. a piece of black paper
18
[Procedure]
1. Take a heavy inoculum, streak for confluent growth on agar
plate, so that the bacteria are evenly and thickly dispersed over
the entire surface of agar plate. Then a piece of black paper is
put on.
2. Half open the plate, treat it with UV light by placing it under
the UV lamp(the UV lamp should be placed about 1 foot from
the cells you wish to irradiate) for 40 mins. Begin timing as
you turn the UV lamp switch on.
3.After irradiation, remove the piece of black paper from the
surface of the agar plate, cover the plate completely, then
invert it, incubate at 37℃for 24 hrs.
4. Observe the results.
(Cheng Yizhe)
19
Exp.6. Effects of chemical disinfectant on
bacteria – paper disk method
[Material]:
slant cultures of staphylococcus aureus, E.coli incubated for 18-24 hr.
0.1% mercury perchloride,
5% crystal violet
2% iodine tincture,
2% red mercury sulfide.
sterile filter (diameter 6.5mm).
nutrient agar plates.
[Procedure]:
1.Take a heave inoculum from the slant culture of S.aureus,
streak on the surface of the agar plate heavily to obtain confluent
growth after incubation. Do the same with the other agar plate,
using the culture of E.coli. Label each plate and divide the plate
into 4 sectors on the bottom, mark the name of each four
disinfectants that you will be testing to each sector.
2. Aseptically pick up a sterile filter paper disk with sterile
forceps, pip the disk into the disinfectant 0.1% mercury
perchloride. Be sure that the excess disinfectant has been drained
off; then place the disk in the center of sector 1 of the S.aureus
inoculated plate. Using the same disinfectant, place another disk in
sector 1 of the E.coli inoculated plate, place the other disinfectants
in the other sectors in the same way.
3. Gently press down the disks with the tip of your flamed
forceps to ensure good contact. Invert the plates, incubate them at
37℃ for 24 hr.
20
4. Next day, observe and compare the inhibition zone around the
disk. Measure the diameter of the zone.
inhibition zone
paper disks
(Cheng Yizhe)
21
Exp.7. Microbial Antibiotic Susceptibility Testing
For some microorganisms, susceptibility to antibiotics is
predictable. However, for many microorganisms (Pseudomonas,
Staphylococcus aureus, and gram-negative enteric bacilli such as
Escherichia coli, Serratia, Proteus, etc.) there is no reliable way of
predicting which antibiotics will be effective in a given case.
Because of many antibiotic-resistant strains of bacteria, antibiotic
susceptibility testing is often essential in order to determine which
antibiotics to use against a specific strain of bacterium.
Several tests may be used to tell which antimicrobial agent is
most likely to combat a specific pathogen:
The agar diffusion test (Bauer-Kirby test)
In the Bauer-Kirby disc diffusion method, a single high-potency
disc of each chosen antibiotics was used. Zones of growth
inhibition surrounding each type of disc were correlated with the
minimum inhibitory concentrations of each antimicrobial agent.
Categories of "Resistant," "Intermediate," and "Sensitive" were then
established.
[Material]
1. Slant agar cultures of Staphylococcus aureus and Escherichia coli
2.Standardized antibiotic-containing discs of antibiotics commonly
effective against gram-positive bacteria, and of antibiotics
commonly effective against gram-negative bacteria
3. Nutrient agar plates
22
[Procedure]
1. Take two agar plates. Label one S. aureus; one E. coli.
2. Using your wax marker, divide each plate into fourth to guide
your streaking.
3. Streak the swab perpendicular to each of S. aureus and E. coli.
drawn on the plate overlapping the streaks to assure complete
coverage of the entire agar surface with inoculum.
4.Using the appropriate antibiotic disc dispenser, place
gram-positive antibiotic-containing discs on the plate of S.
aureus and gram-negative antibiotic-containing discs on the
plates of E. coli.
5.Touch each disc lightly with sterile forceps to make sure it
adheres to the agar surface.
6. Incubate the 2 plates upside-down at 37°C for 24 hors.
7.Using a metric ruler, measure the diameter of the zone of
inhibition around each disc on each plate in mm by placing the
ruler on the bottom of the plate (see fig 8).
8.Determine whether each organism is susceptible, intermediate,
moderately susceptible or resistant to each antibiotics using the
standardized table (Table 2) and record your results.
Fig.8 Measure the diameter of the zone of inhibition
23
The term intermediate generally means that the result is
inconclusive for that drug-organism combination. The term
moderately susceptible is usually applied to those situations where a
drug may be used for infections in a particular body site, e.g.,
cystitis because the drug becomes highly concentrated in the urine.
Table 2. Zone Size Interpretive Chart for Bauer-Kirby Test
antimicrobial
disc
R
I
MS
S
agent
code
= mm or less
= mm
= mm
= mm or more
penicillin
P-10
28
-
-
29
streptomycin
S-10
11
12～14
-
15
chloramphenicol
C-30
12
13～17
-
18
gentamicin
GM-10
12
13～14
-
15
erythromycin
E-15
13
14～22
-
23
tetracycline
Te-30
14
15～18
-
19
SXT
10
11～15
-
16
sulfamethoxazole
+ trimethoprim
[Results]
Interpret the results following steps 7 and 8 of the procedure
and record your results.
Tube Dilution Tests
1. In this test, a series of culture tubes are prepared, each
containing a liquid medium and a different concentration of a
antibiotic.
2. The tubes are then inoculated with the test organism and
incubated for 18-24 hours at 37℃.
24
3. After incubation, the tubes are examined for turbidity (growth).
The lowest concentration of antibiotic capable of preventing
growth of the test organism is the minimum inhibitory
concentration (MIC).
4.Subculturing of tubes showing no turbidity into tubes
containing medium but no antibiotic can determine the
minimum bactericidal concentration (MBC). MBC is the lowest
concentration of the chemotherapeutic agent that results in no
growth (turbidity) of the subcultures.
5. Determine whether each organism is susceptible or resistant to
each antibiotics using the standardized table (Table 3) and
record your results.
These tests, however, are rather time consuming and expensive
to perform.
Table 3.
MIC Interpretive Chart for Tube Dilution Test
antimicrobial agent
R = μg/ml or
more
S =μg/ml or less
penicillin
0.2
0.1
streptomycin
15
6
chloramphenicol
25
12.5
gentamicin
8
4
erythromycin
8
2
tetracycline
16
4
8/152
2/38
sulfamethoxazole +
trimethoprim
(Luan Yi)
25
Exp.8.
Bacteriophages
Content
1. bacteriolysis of bacteriophage
2. bacteriolysis plaque count
Bacteriophages are viruses that infect only bacteria. In addition
to the nucleocapsid or head, some have a rather complex tail
structure,used in adsorption to the cell wall of the host bacterium.
Most bacteriophages replicate by the lytic life cycle and are
called lytic bacteriophages. They can cause osmotic lysis of the
bacterium.
Bacteriolysis of Bacteriophage
[Materials]
1. Slant agar cultures of Shigella flexneri3a and Escherichia coli
2. Bacteriophage of Shigella flexneri3a.
3. Nutrient agar plates.
[Procedure]
1. Divide the Nutrient Agar plate into two parts by a grease
pencil on the bottom.Then label with bacterial names.
2. Streak the two species of bacteria over each helf surface of
the plate with a sterile loop.(make sure to paint each whole
area).
3. When the surface of the agar is DRY, after 3-4 minutes, use
26
the sterile pipet to place one drop of the bacteriophage
solution in the center of each half of the plate.
4. Allow the drop to soak into the agar a few minutes before you
place the plate in the 37℃ incubator. DO NOT INVERT
THESE PLATES.
[Results]
NEXT day, observe the plate and you will see a clear area in
Shigella flexneri3a on there phage added
Bacteriolysis Plaque Count
[Materials]
1.Broth culture of Shigella flexneri 3a Bacteria, suspension of
Bacteriophage of Shigella flexneri3a.
2. Nutrient agar plates, broth,
3. Bottle of sterile saline, sterile 10.0 ml and 1.0 ml pipettes and
pipette fillers, sterile empty dilution tubes,
4. Bottle of melted culture medium from a water bath held at 47℃.
[Procedure]
1.Take 6 tubes containing 9.9 ml or 9.0 ml of sterile saline as
described below and shown in Fig 9.
2.Dilute the Bacteriophage of Shigella flexneri3a stock as
described below and shown in Fig 9.
3. Using a new 1.0 ml pipette to remove 0.1 ml of the
10-5,10-6,10-7, 10-8, bacteriophage dilution respectively to a
new tube, and mix into 0.1 ml of the 10-5,10-6,10-7, 10-8.
4. Using a new 1.0 ml pipette, add 0.1 ml of broth of Shigella
flexneri3a Bacteria to the bacteriophage in each of the 4 tubes
from step 3 and mix.
27
Fig. 9: Dilution of phage for the Plaque Count Experiment
5. Using a new 10.0ml pipette, add 2.5 ml of sterile, melted and
cooled till 47～49℃,culture medium to the bacteria-bacteriophage
mixture in each of the 4 tubes from step 3 and mix from step 4.
6. Quickly pour the motility medium-bacteria-bacteriophage
mixtures onto separate nutrient agar plates and swirl to distribute
the contents over the entire agar surface.
7. Incubate the 3 plates right-side-up at 37°C 24hs.
[Results]
The result is a clear area, or plaque, occurring for each phage
present in the original dilution. By counting these plaques,
multiplying by the dilution factor, the number of phages in the
original material can be calculated. Large excess of bacteria is
necessary so that each cell is infected by only one phage.
(Luan Yi)
28
Exp.9.
Bacterial Variation
I. H-O variation
Bacterial of proteus form a special spreading growth
(swarming or H-growth) on nutrient agar plate because of
having flagella. If the nutrient agar plate contains 0.1% carbolic
acid, the formation of flagella are inhibited, therefore ,instead of
the special spreading growth isolated colonies(O colony)are
formed.
[Materials]
1. broth culture of proteus vulgaris.
2. Nutrient agar plates, 0.1% carbolic agar plate.
[Procedure]
1. Inoculate proteus vulgaris in the centre of the agar plate and
the carbolic agar plate ,do not streak the inoculum,forming a
point of bacteria.
2. After incubation the at 37°C for 24 hours, different results are
shown:
[Results]
(1) On common agar plate，forming spreading growth.
(2) On carbolic agar plate，no spreading growth; Bacteria only
grow around the inoculum.
II. S-R variation
When smooth colony becomes rough colony, the changes are
developed on not only colonies ,but also bacteria toxicities,
biochemical reaction and antigenicities.
[Materials]
1. broth culture of Shigella Sonni (containing bacteria of smooth
colony and rough colony).
2. Nutrient agar plates.
29
[Procudure]
Streak the inoculum on the plate. After incubation the at 37°C
for 24 hours, two kinds of colonies will be seen on the plate.
[Results]
(1) Smooth colony : smooth and moist surface ,tide edge.
(2) Rough colony: rough and dry surface, untidy edge.
III. Bacterial L form variation
For most bacterial cells, the cell wall is critical to cell survival,
yet some bacteria may mutate or change because of extreme
nutritional conditions to form a cell wall-less form or L-forms.
This phenomenon is observed in both G+ and G- species. L-forms
have a varied shape and are sensitive to osmotic shock. The
pathogenicity of bacterial L-forms and their sensitivity to
antimicrobial agents have been different from their protobacteria.
The colonies of spheroplast-L-forms in plate are small and have a
characteristic “fried egg” appearance.
IV. Conjugation transferring of bacterial R plasmid
Many gram-negative bacteria possess R (resistance) plasmids
which have genes coding for multiple antibiotic resistance
through the mechanisms stated above, as well as transfer genes
coding for a sex pilus. Such an organism can conjugate with other
bacteria and transfer an R plasmid to them. Escherichia coli,
Proteus, Serratia, Salmonella, Shigella, and Pseudomonas are
examples of bacteria which frequently have R plasmids.
[Materials]
1. Escherichia coli resistant to ampicillin; Escherichia coli
sensitive to ampicillin; Shigella flexneri sensitive to
ampicillin;
2. LB liquid culture , plates of SS agar with ampicillin
30
[Procudure]
1. Incubate three bacteria into LB culture tubes respectively, at
37℃ for 10 hours.
2. Using a sterile 1ml transfer pipette, mix Escherichia coli
resistant to ampicillin and Shigella flexneri sensitive to
ampicillin or mix Escherichia coli sensitive to ampicillin and
Shigella flexneri sensitive to ampicillin at rate of 1:5
respectively. Incubate at 37℃ for 3 hours.
3. Incubate above three bacteria and two mixed bacteria into
plates of SS agar with ampicillin respectively, at 37℃ for 24
hours.
[Results]
Bacteria in liquid culture
E coli resistant to
ampicillin
E coli sensitive to
ampicillin
S flexneri sensitive to
ampicillin
SS/amp plates
Big red colonies
mixture of E coli resistant
to ampicillin and S flexneri
sensitive to ampicillin at
rate of 1:5
Big red colonies,
and colorless
colonies
Mixture of E coli sensitive
to ampicillin and Shigella
flexneri sensitive to
ampicillin at rate of 1:5
No colony
conclusion
No colony
No colony
R plasmid
conjugation
(Luan Yi)
31
Exp.10.
Bacterial Pathogenicity
Toxicity of tetanus toxin and protection by antitoxin
The elaboration of exotoxins plays a major role in the pathogenesis
of several groups of Gram-positive bacilli.
The clostridia are Gram-positive sporulating bacilli, all of which
are obligate anaerobes. Clostridium tetani are capable of producing
exotoxins---a potent neurotoxin at the site of bacterial replication,
usually in a wound contaminated with fecal material. The exotoxin
binds to ganglioside receptors on inhibitory neurones in CNS and
stops nerve impulse transmission to muscle leading to spastic
paralysis. Continued severe muscle contractions and spasms result
which can be fatal.
[Material]:
1. Two mice: No1 and No2
2. Diluted tetanus toxin, 1:100 diluted tetanus antitoxin
3. Sterile syringes (1 ml)
[Procedure]:
1. Give an injection intraperitoneally of 0.5ml antitoxin to mouse No1.
2. 30 minutes later, inject intramuscularly in back legs of
Mouse1 and Mouse2 respectively of 0.2 ml tetanus toxin.
3. Observe any symptoms that may appear in the two mice after
injection.
[Result]:
Several hours ( about 7 hours) later, the mouse injected only
with toxin may show muscle spasm, the stiffness of leg injected ,
then systemic rigidity, opisthotonus. It will die at about 12hours
after injection, but the other mouse injected with antitoxin before
toxin has no symptom.
(Luan Yi)
32
Exp.11.
Bacterial Plasmid DNA Extraction
Plasmid DNA extraction is the preparation of highly quality
plasmid from liquid culture of bacteria. The obtained plasmid can
be used directly for PCR, restriction enzyme digest or sequencing.
To prepare plasmid DNA can be used by alkaline lysis method,
boiling method et al.
After addition of EDTA and ,in some cases,lysozyme, the cells
are exposed to lyse by treatment with alkali. These treatment ,which
disrupt base pairing ,cause the linear chromosomal DNA of the host
to denature.
Alkaline lysis method
[Materials and Reagents]
1. Ecoli:JM109( including pBR322: Ampr,Tetr)
2. LB media: 3 capsules LB-Medium (BIO 101, Inc)/ 750 ml
H20 in 1 liter bottle. Autoclave 30 minutes, standing on the
rack with plenty of air around the flask. Cool, tighten cap on
bottle, store room temperature.
3. LB-Amp Plates: Ampicillin freezer stock = 50 mg/ml (filter
sterilized in >1ml aliquots); When LB media cool enough to
pour: add 1 ml of amp stock / 500 ml media (100 mg/liter)
and mix well before pouring.
4. GET (50 ml): (store 1.3 ml aliquots at -20°C)
50 mM glucose, 10 mM EDTA, pH 8, 25 mM Tris Cl, pH
8, 2 mg/ml lysozyme, 2 mg/ml RNase A; Dissolve the
RNaseA in approximately 50 ml dH2O, boil for 15 min to
inactivate DNases - being careful not to boil off all the water,
then let cool slowly to room temperature. Add the remaining
33
components and q.s. to 50 ml.
5. Alkaline SDS: (make fresh for each prep)
1 N NaOH 1 ml;
20% SDS 0.5 ml;
dH2O 8.5 ml;
Total Vol 10 ml.
6. High salt solution:
5 M KAc, pH 4.8: 147.3 g KAc,
115 ml glacial acetic acid,
dH2O to 500 ml.
7. phenol
8. chloroform
9. 5mol/L NaCl or 3mol/L NaAc
10. Cold pure ethanol; 70% ethanol;
11. TE buffer: 10 mM Tris-Cl, pH 7.5 ;1 mM EDTA, autoclave.
12. Spin column and EP Tube, Store at room temperature.
[Procedure]
I. Growing the cell culture.
1. Inoculate E. coli （with pBR322 plasmid）cells overnight at
37℃ in 5 ml LA medium.
2. Patch a small amount to a fresh LA plate and grow overnight
at 37℃, as well.
3. Inoculate a single colony of E. coli in 2ml sterile LA medium,
at 37°C overnight in a shaker water bath.
4. Inoculate E. coli in 50ml sterile LA medium, at 37°C in 3~4h
in a shaker water bath till OD600=1.0.
II. Preparation of the cell lysate
1. Fill a microfuge tube with the culture and pellet cells by
spinning in microcentrifuge for 2 minutes.
34
2. Decant supernatant and resuspend cell pellet in 1ml GET
solution. Spin for 2 minutes again.
3. Decant supernatant and resuspend cell pellet in 80µl GET
solution. Add 25µl of freshly prepared lysozyme (finally to
2mg/ml). Mix by shaking gently.
4. Place the tube in a boiling water bath for 60 seconds. Cool on
ice for 5 minutes.
III. plasmid DNA extraction and purify
1. Add 200 µl alkaline SDS, mix by repeated inversion. Incubate
5 min at room temperature.
2. Add 150 µl high salt solution and MIX WELL. Incubate 30
minutes on ice.
3. Spin in 12000rpm microfuge for 10 min.
4. Transfer liquid 400 µl to a fresh tube, trying to avoid floating
particles as much as possible.Add 200 µl phenol and 200 µl
chloroform, mix 5 min. Spin in 12000rpm microfuge for 15 min.
5. Transfer liquid to a fresh tube, add 200 µl, mix 5 min. Spin in
12000rpm microfuge for 15 min.
6. Transfer liquid to a fresh tube, add 1/20 volume NaAc, then add
cold 2 volumes ethanol, mix and place under -20℃ overnight.
7. Precipitate nucleic acids with spinning in 12000rpm
microfuge for 15 min.
8. Remove supernatant. Add 1 ml cold 70% ethanol. Spin in
12000 rpm microfuge for 15 min.
9. Repeat step 8. Remove supernatant. Dry pellet briefly at room
temperature.
10. Resuspend pellet in 50 µl TE. Store the DNA at -20℃.
11. 2 µl plasmids DNA is sufficient for analysis on a mini-gel.
(Luan Yi)
35
Exp.12.
Pathogenic Cocci
Content:
1. Morphology
2. Culture characteristics
3. Coagulase test
4. Identification of pathogenic cocci from clinical specimen
Morphology
Observe Gram stain slides under microscope of Staphylococci,
Streptococci, Streptococcus pneumonia, Neisseria meningitides.
Pay attention to their shape, stain properties and arrangement.
Culture Characteristics
Observe colonies of S.aureus, S.epidermis, α -hemolytic
streptococci, β -hemolytic streptococci, Strep. Pneumoniae on
common agar plate and on blood agar plate. Note their shape,
surface, margin, transparency, hemplysis and pigment. Compare
colonies of S. epidermis with S. aureus, α-hemolytic streptococci
withβ-hemolytic streptococci, S. pneumoniae withα-hemolytic
streptococci.
36
Coagulase Test
Coagulase is an enzyme that causes the fibrin of blood plasma
to clot, pathogenic staphy. Aureus produces coagulase, while
non-pathogenic strains are coagulase negative.
[Material]:
1. Slant and broth cultures of Staphy. Aureus and staphy. Epidermidis
2. Rabbit plasma or human plasma
3. Glass slide, test tube, NS
[Procedure]:
1. Slide method ( testing for bound coagulase ):
(1) Place a drop of NS on a slide.
(2) Emulsify the organism to be tested in the drop of NS.
(3) Place a drop of plasma immediately adjacent to the
drop of the bacterial suspension and mix thoroughly.
(4) While tilting the slide back and forth, observe the
immediate formation of a granular precipitate of white
clumps.
(5) Practice control in the same way but without plasma.
2. Tube method ( testing for free coagulase ):
(1) Add 0.5ml of plasma to the bottom of each of 2 test tubes.
(2) Add 0.5ml of 24 hrs pure broth culture of the organism to be
tested in one tube, and 0.5ml of blank medium to the other.
(3) Mix by gentle rotation of the tube, avoid stirring or
shaking of the mixture.
(4) Place the tube in 37℃water–bath for 1.5～2hrs. Observe
for formation of visible clot. Compare results in the 2 tubes.
37
[Interpretation]:
1.Slide method: A positive reaction is usually detected within
15-20 secs by the appearance of a granular precipitate or
formation of white clumps. The test is considered negative if
clumping is not observed within 2～3mins. The slide test is
considered only presumptive, all cultures giving negative or
delayed positive results should be checked with the tube test
because some strains of S. aureus produce free coagulase that
does not react in the slide test.
2. Tube method: The reaction is considered positive if any degree
of clotting is visible within the tube. The test is best observed by
tilting the tube. The clot or gel will remain in the bottom of the
tube if the test is positive for coagulase.
Strong coagulase-positive bacteria may produce clot within
1～4hrs; therefore, it is recommended that the clot be observed
at 30mins intervals for the first 4 hrs of the test. Strong
fibrinolysins may also be formed by some S. aureus strains and
may dissolve the clot soon after it is formed.
Therefore, positive tests may be missed if the tube is not
observed at frequent intervals. Other S. aureus strains may
produce only enough coagulase to produce a delayed positive
result after 18～24hrs of incubation. Therefore all negative tests
at 4 hrs should be again observed after 18-24hrs of incubation.
38
Identification of Pathogenic Cocci from Clinical Specimen
[Material]:
1. Specimen: pus collected from wound
2. Media: blood agar plate, mannitol fermentation tube
3. Rabbit plasma ( or human plasma )
[Procedure]:
Pus→Gram strain→Microscopic examination
↓
Streak on→Gram strain→Colonies observation
↓
Biochemical reaction
Pathogenicity test
1. Direct smear:
Make a smear with pus, stain with Gram reagents,
examine under microscope, note the Gram stain property,
shape and arrangement of bacteria if present.
2. Isolation: streak the pus on a blood agar plate for isolation,
incubate the plate at 37℃ for 18～24hrs.
3. After incubation, observe colonies and their hemolytic properties.
(1) Stain with Gram reagents, examine under microscope;
(2) Examine for biochemical reaction, e.g. mannitol
fermentation, and pathogenicity, e.g. coagulase test.
(Qi Mei)
39
Exp.13.
Enteric Bacilli
Content:
1. Morphology
2. Media for cultivation of Enteric bacilli
3. Biochemical reaction
4. Identification of intestinal pathogens feces
Morphology
Under microscope, observe Gram stain slides of E.coli,
Salmonella typhus, shigella.
Media for Cultivation of Enteric Bacilli
1. Selective medium
These media incorporate chemical substances that inhibit the
growth of one type of bacterial while permitting growth of another,
thus facilitating bacterial isolation. S-S agar plate
( Salmonella-Shigella agar plate ), is selective medium used for
isolation of Salmonella or Shigella.
2. Differential medium
These can distinguish among morphologically and
biochemically related groups of organisms. They incorporate
chemical compounds that, following inoculation and incubation,
produce a characteristic change in the appearance of bacterial
growth and/or the medium surrounding the colonies, which
permits differentiation. For example, EMB agar (eosin methylene
blue agar ) contains lactose and eosin methylene blue dyes which
40
permits differentiation between enteric lactose fermenters and
nonfermenters, the E.coli colonies are blue-black with metallic
green sheen caused by large quantity of acid that is produced and
precipitates onto the growth surface. Pathogenic bacteria that do
not ferment lactose produce colorless colonies, which because of
their transparency, appear to take on the purple color of the
medium. This medium is also partially inhibitory to the growth of
Gram positive organisms, thus gram-negative growth is more
aboundant.
3. Double sugar iron agar slant
The upper part is solid, contains lactose and FeSO4, phenol red
as indicator; the lower part is semisolid, contains glucose, phenol red.
Purpose:
(1) Identify pathogenic or non-pathogenic enteric bacilli by
testing their lactose and glucose-fermenting ability;
(2) Obtain pure culture by streaking on the slant a single
colony from a primary or selective agar plate;
(3) Observe gas-production and motility through the lower part;
(4) Detect H2S formation; if bacteria produce H2S, black
precipitate will appear in upper part.
Biochemical Reaction
1. Carbohydrate fermentation:
Many bacteria have the ability to ferment a wide range of
carbohydrates, the pattern of fermentation is characteristic of
certain species, genera, or other taxonomic group. Reaction
concerning carbohydrates fermentation by one particular
organism may lead in the identification of the organism.
To study the physiological differences of bacteria, you will be
using fermentation tubes. A fermentation tube is a culture tube
41
that contains the following:
(1)Durham tube: A small tube that is placed upside down in the
culture tube. The purpose of the inverted Durham tube is to show
if gas has been produced. This does not allow you to determine
which gas is being involved. The most common gases produced by
bacteria are O2, CO2, and methane.
(2)pH indicator: A medium that contains the ingredients of
nutrient broth, plus a pH indicator. The nutrient broth supports the
growth of most bacteria. The commonly used pH indicator is
bromocresol purple, which appears purple at a pH near neutral,
and turns yellow at an acid conditional
(3)A specific carbohydrate substrate: Such as glucose,
lactose, maltose, mannitol, or sucrose. If a single carbohydrate is
fermented, an acid environment results, and the broth turns
yellow. Gas, if produced, will be trapped in Durham tube.
There are three types of carbohydrate fermentation:
－: negative reaction
＋: acid production
♁: acid and gas production
2. Sulfide ( H2S ) production:
Many organisms manufacture enzymes necessary to liberate
H2S from S-containing organic compounds ( such as cystine or
cysteine ). When H2S is liberated, it reacts with iron sulfate or
lead sulfide. They are manifested as black insoluble precipitate
causing extensive blackening of the culture.
3. Indole production:
Some bacteria possess the enzyme tryptophanase and
decompose tryptophane, resulting in the production of indole
which accumulates in the medium. Indole can be detected in a
medium as reagent. The active component of Kovac's reagent is
p-dimethylaminobenzal dehyde which reacts with indole to
produce a cherrhy compound-rose indole.
42
4. Urea hydrolysis:
Some organisms possess the enzyme urease which can
hydrolyse urea with the production of ammonia. The alkaline pH
can be detected by an indicator.
This test is performed by inoculating a urea medium with the
test organisms and inocubating it at 37℃, for18-24hrs. A change
in color of the medium (with phenol red )from buff to bright
pink represents a positive result, on change in color shows a negative
result.
Identification of Intestinal Pathogens from Feces
[Material]:
1. Specimen: patient's feces or rectal swab
2. Media: EMB agar plate, SS agar plate, double sugar iron agar slant
3. Diagnostic sera: monovalent antisera and polyvalent antisera
against Salmonella and Shigella
[Procedure]
1. Streak the feces or rectal swab on EMB and/or S.S agar
plate for isolation. Inocubate at 37℃ for 24hrs.
2.Take the grey white single colonies ( lactose-nonfermenting colony )
stab in double sugar iron agar and then streak on the slant
for pure culture, incubate at 37℃ for 24hrs.
3.Observe bacteria growth characteristics in/on the double
sugar iron Slant. According to the growth pattern, a
presumptive diagnosis can be made.
4.Slide agglutination of the isolated pathogen with specific
antisera and sugar fermentation tests should be done for
further identification.
43
5.Steps for identification are summarized as follows:
Specimen ( feces or rectal swab )
↓
Streak on EMB or SS agar plate
↓
Streak on double sugar iron agar medium
↓
Agglutinate with polyvalent antisera against
Salmonella & Shigella
↙
↘
Biochemical reaction
Agglutinate with monovalent
antisera of Salmonella &
Shigella
6.Growth patterns of different specles or enterobacteria are
shown in the following table. (in double sugar iron agar
medium )
Table 4. Fermentation of bacteria in double sugar iron agar medium
Upper
Lactose
part
lower
part
H2S
glucose
motility
±
♁
+
E.coli
♁
S.typhi
-
-
-
+
shigella+-
-
-
+
(Qi Mei)
44
Exp.14.
Clostridium
Content:
1. Morphology
2. Anaerobic cultivation
3. Animal model of Clostridium perfringens infection
Morphology
Under microscope, observe the slides ( endospore stain ) of
Cl.tetani, Cl.perfringens, CL.botulinum. Note the bacteria
shape, endospore site and position.
Anaerobic culture
1. Observe colonies of Cl.tetani on blood agar plate, note
their shape and hemolysis.
2. Observe growth characteristics of Cl.tetani and
CL.perfringens in beef media.
Animal Test of Cl.perfringens
[Material]
Culture of Cl.perfringens in beef broth, mouse, syringe
[Procedure]
1. Drain off culture of Cl.perfringens 0.2～1.0ml, inject
intravenously into the caudal vein of a mouse.
2. Kill the mouse after 5 mins, then incubate at 37℃ for 5～8hrs.
3. Observe the swelling phenomenon in the dead mouse,
dissect the animal, note the terrible odor spreading from it
and the gross pathologic changes.
4. Make direct smear from the liver, lood for the pathogen
after Gram stain.
(Qi Mei)
45
Exp.15.
Mycobacterium
Content:
1. Morphology
2. Culture characteristics of M.tuberculosis
3. Isolation of M.tuberculosis from sputum
Morphology
Under microscope, observe acid-fast stain slides of M.tuberculosis
and M.leprae.
Culture Characteristics of M.tuberculosis
Observe colonies of M.tuberculosis on Trudeau's agar slant.
Isolation of M.tuberculosis from Sputum
[Material]
1. Sputum from open case of pulmonary tuberculosis
2. Acid-fast stain reagents: carbolfuchsin, acid alcohol,
methylente blue.
[Procedure]
1. Make a smear of the sputum, dry and fix it.
2. Ziehl-Neelsen acid-fast stain:
(1) Flood the slide with carbolfuchsin. Heat the slide to
steaming for 5mins. Do not boil or allow the smear to dry. As
stain evaporated from the slide, replenish with additional
carbolfuchsin. Allow the slide to cool and rinse it thoroughly
with water.
(2) Decolorize the slide with acid alcohol until the red color
no longer comes off in the decolorizer. It takes about 30secs.
Rinse the slide with water.
(3) Counterstain with methylene blue, allow the stain to react for
1min, rinse as above.
(4) Bolt the slide carefully. Examine under microscope.
(Qi Mei)
46
Exp.16.
Polymerase Chain Reaction ( PCR )
[Principle]:
The polymerase chain reaction ( PCR ) is a rapid technique
for the enzymatic in vitro amplification of DNA or RNA by
which a particular segment of nucleic acids can be specifically
replicated.
It involves two oligonucleotide primers that flank the DNA
fragment to be amplified and repeated cycles of heat
denaturation of the DNA, annealing of the primers to their
complementary sequences, and extension of the annealed
primers with DNA polymerase. These primers hybridize to
opposite strands of the target sequence and are oriented so that
DNA synthesis by the polymerase proceeds across the region
between the primers. Since the extension products themselves
are also complimentary to and capable of binding primers,
successive cycles of amplification essentially double the amount
of the target DNA synthesized in the previous cycle. The result
is an exponential accumulation of the specific target fragment,
approximately 2n, where n is the number of cycles of
amplification performed.
[Material]
1. Taq DNA polymerase and Enzyme concentration
( 1～2.5u/per 100μl reaction ).
2. Deoxynucleotide triphasphotes (dNTP )
3. Magnesium concentration ( 0.5～2.5nM )
4. Other reaction components:
① PCR buffer ( 10～50nM tris-HCL, pH8.3～8.8 )
② 50mM KCl
③ DMSO
[Procedure]
47
1. primer annealing ( 55-72℃ )
2. primer extension ( 72℃ )
3. Denaturation time & temperature ( 95℃, 15～30secs; The
half-life of Tag DNA polymerase activity is >2hrs, 40mins,
and 5 mins at 92.5, 95,and 97.5℃, respectively )
4. Cycle number:
No. of
Target Molecules
3×105
1.5×104
1×103
50
No. of cycles
25-30
30-35
35-40
40-45
5. Primers
①General primer concentration (0.1～0.5μM)
② Higher primer concentration-promote mispriming and
accumalation of nonspecific product (“primer-dimer”).
③Typical primers length (18-28nucleotides 50～60%G+C)
[Standard PCR amplication protocol]
1. Set up a 100μl reaction in a 0.5ml microfuge, mix, and
overlay with 75μl of mineral oil.
Template DNA(105-106 target molecules)
20pM each primer(Tm>55℃ preferred)
20mM Tris-HCl(pH8.3)(20℃)
5mM KCl
0.05% Tween-20
100μg/ml(autoclaved gelatin or nuclease-free bovine)
Serum albumin
50μM each dNTP
2 units of Taq DNA polymerase
48
2.
3.
4.
5.
Note:
1μg of human single-copy genomic DNA=3×105 targets;
10ng of yeast DNA=3×105 targets;
1ng of E.coli DNA=3×105 targets;
1% of M13plaque=106 targets.
Perform 25 to 35 cycles of PCR using the following
temperature profile:
Denaturation 96℃, 15seconds.
Primer annealing55℃ ,30seconds.
Primer extension 72℃,1.5mins.
Cycling: Conclude with a final extension at 72℃ for 5mins.
Stop reaction (chilling to 4℃and/or by addition of EDTA to 10mM).
Electrophoresis: Assay MW of amplicated target nucleic acid.
[Applications]:
1.Research Applications:
(1) Sequencing with Taq DNA Polymerase
(2) Amplification of Flanking sequence by inverse PCR
(3) Detection of Homologous Recombinants
(4) Recombinant PCR
(5) In vitro Transcription of PCR Templates
(6) Screening of Lambda λgt lilbraries
2.Genetics and Evolution
(1) HLA DNA typing
(2) Amplification of ribosomal RNA genes for molecular
evolution studies
(3) Haplotype analysis from single sperm or diploid cells
(4) Amplification & direct sequencing of ribosomal RNA genes
3.Diagnostics & Forensics
49
(1) Detection of Human T-cell lymphoma/leukemia viruses
(2) Detection of Human Immunodeficiency virus
(3) Detection of HBV, HPV, HCMV, Enterovirus, novel viruses
(4) Analysis of ras gene point mutations by PCR and
oligonucleotide hybrization
(5) B-cell lymphoma-chromosome rearrangement
(6) Detecting bacterial pathogen
Determination of familial relationships
(Qi Mei)
Exp.17.
Mycoplasma
1. Morphology
In Giemsa stain slide, membrane---bounded cells appear
as coccoid bodies filamentous, and star—shaped forms. The
size of individual organisms can be approximated as spheres of
0.3-0.5μm in diameter.
Draw down a microscopic view of the slide.
2. Culture characteristics
Organisms form small colonies, 10～100μm in diameter, on
enriched medium, colonies frequently show “fried-egg”
appearance (colony shoes a dark center where organisms have
grown into the agar medium).
Very faint turbidity is produced in broth culture.
(Zhou Yabin)
50
Exp.18.
Rickettsiae
1. Morphology
In Giemsa stain slide, observation with microscope: usually as a
purple bipolar rod, near cell or in cytoplasma.
Draw down a microscopic view of the slide.
2. Weil-Felix test
[Principle]
The test depends on the agglutination of OX19, OX2, and
OXk stain of Proteus vulgsris by Abs produced by rickettsiae.
Proteus organisms have nothing whatever to do with causing
rickttsiae disease, but certain strains of bacteria have an Ag on
their cells that is also present in the rickttsiae. The test is a
valuable aid in diagnosis.
[Material]
1. Patient’s serum
2. Ag: P. OX19 suspension
[Procedure]:
1. Dilute serum as the following table and then add Ag (OX19)
2. Shake thoroughly, incubate at 37 overnight.
3. Next day, observe agglutination and determine the serum titer.
Table 5. Procedure of Well-Felix Test
Number of
1
2
3
4
5
6
7
8
9
10
Dilution of
1:1
1:2
1:4
1:8
serum
0
0
0
0
1:160
1:320
1:640
1:1280
1:2560
NS
Serum (ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Ag (ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
tube
(Zhou Yabin)
51
Exp.19.
Chlamydia
Observe the characteristic inclusion bodies: located in
cytoplasma of conjunctival cells, appearing basophilic, with
irregular shapes (compact-type, cap-type, diffusion-type,
mulberry-type, spreading-type).
Draw down a microscopic view, which presents cells containing
inclusion bodies.
(Zhou Yabin)
Exp.20.
Spirochetes
I. Stain slide observation:
1. Leptospira: appearing brown in silver-deposition
stain, exhibiting a fine and tightly coiled spiral,
usually possessing a hook at one or both ends.
2. Borrelia recurrentis: Wright stain B. recurrentis in the blood
smear of patient in fever, the Borrelia appear dark-red,
filamentous spiral-shaped.
3. Treponema pallidum: silver-deposition stain of T. pallidum
in the liver slice of patient, exhibiting brown spiral-shape.
II. Examination of Borrelia vincentii from odontolite:
[Material]
Sterile tooth pick
Fix solution, mordant stain, and silver nitrate stain
[Procedure]
1. Add 2～3 loops of NS on a slide;
2. Take a little odontolite with tooth pick, mix with the NS,
make a smear;
52
3. Add fix solution on the dried slide, react for 1 min, and
then rinse with water.
4. Add mordant solution, stain for 30 seconds, rinse again;
5. Silver nitrate stain for 30 seconds, rinse again;
6. Dry with didulous paper, examine with microscope
spirochetes are stained brown. Spirochetes are stained
brown.
III. Observation of Leptospira under dark-field microscope:
Add a drop of Leptospira fluid culture on a slide, and then put
a cover slip on. Observe under dark-field microscope, note their
shape and movement.
(Zhou Yabin)
Exp.21.
Actinomycetes
I.Observe H-E stain slide from infected tissue.
Sulfur granule formd by A. Bovis in the slide of infected tissue,
note the peripheral “clubs”.
II.Observe culture and slide of Actinomycetes
note the morphology of the colonies, hyphae and external spores.
(Zhou Yabin)
53
Exp.22.
Fungi
I. Morphology of Fungi
1. Unicellular fungi:
Candida
albicans-----note
clamydospores,
blastopores
and
pseudohyphea under microscope.
2.Multicellular fungi-----observe following properties under
microscope:
Hyphae;
Asexual spore: blastospore,
clamydospore,
arthrospore,
sporangiospore,
microconidium,
macroconidium;
II. Culture of Fungi
1. Medium:
Sabouraud’s dextrose agar is commonly used medium for
cultivation of fungi.
The composition is:
dextrose
40g
peptone
10g
agar
15g
distilled water
2L
Final pH=5.6
2. Colony types:
(1) Yeast-form colony: e.g. Yeast; Cryptococcus neoformans.
(2) Yeast-like colony: e.g. Candida albicans.
(3) Hyphomycete-form colony: e.g. Dermatophytes.
54
3. Microslide cultivation technique:
[Material]
(1) Fungi culture on Sabourad’s medium or potato dextrose agar.
(2) Slide, coverslip.
[Procedure]
(1) Place a block of Sabouraud’s dextrose agar (about 1cm2)
on a sterile slide.
(2) Using a straight inoculating needle, inoculate the
specimen to be assayed on each side of the block.
(3) Gently heat a coverslip by passing it quickly through the
flame of an alcohol burner and immediately place it
directly on the surface of the inoculated agar block.
(4) Place the slide in a petri dish on a slide supporter glass
rods above a moistened filter paper disk, incubate at 28℃
for 3～5 days.
(5) The slide can be microscoped at intervals during
cultivation to observe the dynamics of hyphae and spore
growth.
4.Specimen examination from superficial fungal infection
[Material]
(1) Infected skin scraping or infected hair.
(2) 10～20% NaOH solution.
[Procedure]
(1) Place the specimen on a slide.
(2) Add a few drop of NaOH solution on the specimen, put on
a coverslip, heat by passing through the flame of alcohol
burner several times, then leave it at room temperature for
10 mins.
55
(3) Examine under microscope, note the hyphae and external spores.
III.Pathogenic fungi observation of deep mycoses
Characteristics of Cryptococcus neoformans:
1. Yeast-form colony;
2. Pathogenic Cryptococcus neoformans grow at 37℃ and 22℃;
3. Negative ink---stain slide of Cryptococcus neoformans;
(1) Add a drop of NS on a slide;
(2) Immerse Cryptococcus neoformans culture in NS;
(3) Add a drop of ink, put a coverslip on it;
(4) Observe under microscope, note the unstained thick
layer of capsule around the fungal cell.
(Zhou Yabin)
Exp.23.
Viral Morphology
1. Observe electron microscope pictures of Adenovirus, Influenza
virus, Herpes virus and Bacteriophage. Note their shape, size,
arrangement and structure.
2. Observe virus inclusion bodies:
Rabies virus: acidophilic Negri bodies in thr cytoplasma of
infected brain cell (located in the brain tissue of
gyrus hippocampi)
Measles virus: acidophilic, irregular inclusion bodies in
cytoplasma and nuclei of cultured human embryo
kidney cell.
(Zhou Yabin)
56
Exp.24.
Culture of Aminal Virus
Content:
1.Culture in chicken embryo
2.Culture in cell
3.Culture in experimental animals
Culture in Chicken Embryo
Chicken embryo culture is the technique commonly used
for virus and rickettsiae cultivation. Some virues, such as
Othomysovirus,
Paramyxovirus,
Poxvirus,Herpetovirus,
Rabiesvirus and viruses inducing encephalitis grow well in
chicken embryo.
1. Inoculation into allantoic cavity
(1) Take a 9～10 day-old chicken embryo, mark the position
of air sac and embryo, put the agg in panallel with the
experimental bench.
(2) Sterilize the location for injection on the egg shell.
(3) Open a small hole on the shell at 0.5 cm above the air sac
and away from the embryo( Fig 11).
(4) Puncture 1 cm deep, inject 0.1～0.2 ml of virus sample.
(5) Seal the hole with sterile plastic, incubate the embryo at
36℃. Gently rotate the embryo twice a day, examine in
egg check box once a day. After incubation for 2～3 days,
harvest the allantoic fluid, detect the presence of virus,
preserve at 4℃ for use.
The method is suitable for culture of some respiratory viruses,
e.g. Influenza virus
57
2. Inoculation into yolk sac:
(1) Take 6～8 day-old chicken embryo, mark it .
(2) After sterilization the shell, open a hole in the center of
air sac ares.
(3) Puncture 3 cm deep, inject 0.5 ml of virus sample.
(4) Seal the holr as above (Fig 11).
(5) Incubate and harvest the yolk membrane which contains
respective virus, preserve at 25℃ for use.
The method is also suitable for Rickettsiae and Chlamydia
trachomatis.
yolk sac
Fig 10. Inoculation into yolk sac
Fig 11. Inoculation into allantoic cavity
Culture in Cell
1. Primary cell culture:
[Material]:
Growth medium
0.5% Lactalbumin in Hanks' s PBS
78 ml
calf serum
10 ml
human serum
10 ml
sodium bicarbonate solution
1 ml
penicillin and streptomycin
1 ml
58
[Procedures]:
Remove the kidneys from suckling mouse using aseptic
precaution throughout (through sections on both sides sides
of the back).Decapsulate the kidneys and dissect sagitally
the medulla and pelvis mince the cortex finely with scissors,
and wash the minced tissue several times in Hanks' solution
to remove blood cell and debris. Add appropriate amount of
0.25% trypsin, trypsinize at 37℃ for 30 min; Discard the
claudy supernate which many contain toxic matter and add
trypsin solusion containing antibiotics. Trypsinize until
tissue disintegrate, forming cloudy suspension of cells.
Filter the suspension through sterile gause and centrifuge
filtrate 10 min, at 1000rpm. Discard supernate and
resuspend in growth medium. Adjust cell concentration to
1.5 × 105 cellses/ mls, dispense into tubes and bottles.
Incubate at 37℃ until monolayered.
2. CPE observation
[Procedures]:
(1) Inoculate a known virus culture (e.g. HSV) into the
monolayered cell culture, incubate at 35℃, observe daily,
note the pathologic changes produced.
(2) Mock infect the cell culture with blank Hanks’ solution,
compare the cells appearance.
59
Culture in Experimental Animal
Mouse brain inoculation:
1. Sterilize the site for injection.
2. Inject 0.1 ml of Japanese B encephalitis virus culture fluid
into the mouse brain.
The method is suitable for inoculation of encephalitis viruses and
some enteric virus.
(Zhou Yabin)
60
Exp. 25.
Viral Serology
Content:
1. Haemagglutination test
2. Haemagglutination-inhibition test
Haemagglutination Test
[Material]:
Virus liquid, 1% chicken RBC (CRBC), microhaemagglutination trays.
[Procedure]:
1.Dilute virus as indicated in the following table. (Table 6 )
2. Add NS and CRBC.
3. After thoroughly mixing, place the trays at room temperature
for 30~40mins.
4. Read the result of heamgglination, the haemagglutination titer is the
highest dilutions, which still cause obvious haemagglutination (++).
Haemagglutination Inhibition Test
[Material]:
Virus(4u), patient’s serum, 1% CRBC
[Procedure]:
1.
2.
3.
4.
Dilute the serum as illustrated in the table (Table 7 )
Add haemagglutinin, NS and 1% CRBC.
Mix thoroughly, then incubate at room temperature for 30~40min.
Observe the result, determine the serum titer, the titer is the
highest dilution that still inhibits haemagglutination completely.
(Zhou Yabin)
61
Table 6. Procedure of Haemagglutination Test
No. Of well
1
2
3
4
5
6
7
8
9(control)
Virus (ml)
1:10
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
-
0.2
0.2
0.2
0.2
0.2
0.2
0.2
-
-
NS (ml)
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.4
CRBC (ml)
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Table 7
No. of
well
1
2
Procedure of Haemagglutination Inhibition Test
3
4
5
6
7
8
9
10
(serum
(virus
control) control)
1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280
1:10
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Virus(ml)
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
-
0.2
NS(ml)
-
-
-
-
-
-
-
-
0.2
0.2
CRBC(ml)
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Serum(ml)
63
-
64