Laboratory Manual of Microbiology
For Medical Students
Department of Microbiology
Gannan medical Univerisity 2002
General Rules for Microbiology Laboratory
The laboratory work of microbiological course involves handing cultures and
materials, which are infectious in nature, for safety in the laboratory, please
observe the following precautions:
1. Wear a laboratory coat before entering the laboratory.
2. No eating, smoking and drinking in the laboratory.
3. Dispose of infectious materials and cultures carefully to avoid endangering
your health. Put the contaminated glassware into the large cylinder with lysol
solution, and the materials to be sterilized or disposed at the indicated area.
4. Be very careful to avoid spilling out any kinds of contaminated materials. If
the infectious materials contaminated the desk, hands, laboratory coat or floor
notifies your instructor at once.
5. Take care of the glassware and instruments you used, in case of any
breakage, report to your instructor.
6. Do not remove specimens, cultures or equipment out of the laboratory
under any circumstances.
7. Keep personal items in the places designated and away from working area.
8. After finishing the experiment, scrub the laboratory tables with cleaning
agent, wash your hands thoroughly, close the windows, and turn off the electric
light and tap water before leaving the laboratory.
Experiment 1 Use and Care of Microscope
One of the most useful tools for Microbiology study is the microscope. It is
assumed that this instrument has been used and is generally familiar with the parts
of lt. For this reason, discussion of the instrument will be restricted to the use and
care of oil immersion lens.
Oil-immersion lens in size is smaller than other lens. The rays of light
undergo refraction by air and pass into the lens very little. The space between the
lens and object is occupied by immersion oil, which has the same refractive index
as the glass; so light pass through the lens is brighter. The objects can be seen
clearly.
A．Materials
light microscope small bottle of immersion oil (cedal oil)
xylene lens paper
prepared practice slides of bacteria
B. Procedure
1. Place microscope in proper working position of the table at convenient
height. Be sure you are comfortably seated and look into the microscope.
2. Adjust the mirror or the illumination, so that you can find the brightest
microscopic field.
3. Put the slide on the stage and place a drop of immersion oil on the slide.
4. Lower the oil immersion objective with the coarse adjustment into the drop
of oil until the objective almost touches the slide but does not touch it.
5. Focus upward with the coarse adjustment knob very slowly until the image
appears while looking into eyepiece.
6. Complete focus with fine adjustment.
C. Care of Microscope
1. When the experiment is over, clean the microscope. Be sure to remove oil
from the oil immersion objective with lens paper moistened with xylene, then use
dry lens paper to remove the xylene on lens.
2. Because the microscope is an expensive and fine instrument, especially the
oil-immersion lens. It is necessary to take care of it. Do not drop and lay it on the
wrong place and be sure to place it in the case.
Experiment 2 Basic Shapes and Special Structures of Bacteria
1.Materials
Smears of Staphylococcus aureus, Escherichia coli and Vibrio cholera, which
are stained by Gram stain. Smears of Streptococcus pneumoniae , Proteus
vulgaris and Clostridium tetani , which are stained by special stain.
2.Procedure
Observe these sears on the microscope, and pay attention to the morphology, the
arrangement and the staining properties.
3. Result
Coccus: Staphylococcus.aureus
Basic shapes:
Bacillus: Escherichia coli
Vibrio: Vibrio cholera
Special structures:
Capsule: Streptococcus pneumoniae
Flagella: Proteus vulgaris
Spore: Vibrio cholera
Experiment 3
Preparation of Culture Media
In order to cultivate the bacteria artificially, it is necessary to provide suitable
nutrients for their growth. Medium is the material prepared artificially in which or
on which the bacteria are grown. The media used for the cultivation of the bacteria
may be liquid, semisolid, or solid. The media that support the growth of a large
number of bacteria are called general-purpose media. Selective media contain some
chemical substances, which can prevent the growth of one, or more groups of
bacteria without inhibiting the growth of desired one. Differentiate media contain
certain chemicals which permit the observer to differentiate the bacteria. The basic
components of media consist of peptone, amino acids, sugars, salts and water.
Besides, the media should be adjusted to optimal pH (usually 7.4-7.6) and be
sterilized.
A. Broth Medium
1. Materials
Finely ground beef
Peptone NaCl K2HPO4 Distilled water
2. Procedure
a. Add 1000ml of distilled water to 500g finely ground lean beef (fascia and
fat are completely removed). Put in the refrigerator over night; let the nutrients in
fuse out from the beef. Remove all the fat on the surface of the infusion liquid.
b. Boil the infusion for 30 min, filter through a piece of cloth, squeeze out all
the liquid from the beef residues.
c. Add distilled water to restore the volume up to 1000ml. Put 10g of peptone
and 5g of NaCl to the infusion.
d. Cool to 40-50℃，adjust pH to 7.8 with NaOH, boil for 10 min and restore
the volume with distilled water, filter through absorbent cotton.
e. Dispense into tubes or flasks, and stopped with cotton plugs.
f. Sterilize in autoclave at 15 lbs (121℃) for 20 min.
Note
If there are not any fresh beef available, we can use the meat extract (meat
infusion) 3g.
B. Solid Medium
Agar is usually used to prepare the solid media. Agar itself doesn’t posses any
nutritive value for the growth of bacteria, but it is used in the media to alter the
physical properties of broth , for it can melt at 100℃ and solidify around 40℃.
Add 2% agar to the broth, we will get a solid medium. If 0.5% agar is added, a
semisolid medium will be formed.
1. Materials
Agar Petri dish
Pipet Broth Medium
2. Procedure
a. Add 2-3 g of agar to 100ml broth in a flask; heat until agar is completely
dissolved.
b. Adjust pH to 7.4-7.6 with NaOH.
c. Sterilize in autoclave at 15 lbs for 20 min.
d. When the medium is still liquid, pour it to the sterile Petri dish, allow it to
cool to 40℃ and agar plate is formed.
5. Pour 4-5ml of medium to sterile tube and let the tube slant, agar slant is
formed.
Note
If add 0.5% agar to the broth, the medium becomes semisolid.
Experiment 4 Examination of Bacterial Metabolic Products
It is common for two bacterial cultures to be very similar in their morphologic
and cultural characteristics, but strikingly different in their ability to use nutrients
and form the end products. These characteristics of bacteria are indispensable in
classifying and identifying the bacteria.
A. Sugar fermentation of bacteria.
Bacteria can utilize different sugars and produce different end products, for
they possess different enzyme. The common method used is a routine qualitative
test designed to permit to detect the sugar fermentation by noting the change in pH
of culture medium. The medium usually consists of certain sugar and indicator,
such as bromcresol-purple, which will change color from purple (pH 7) to yellow
(pH 5.4), if the bacteria ferment the sugar and produce acid. If gases are produced,
they will trap in the small inverted vial in the tube.
1. Materials
Slant cultures of E. coli and S. Typhoid
Wire loop
Glucose and lactose fermentation tubes Alcohol lamp
2. Procedure
a. Inoculate two bacterial cultures into fermentation tubes, respectively.
Incubate at 37℃ for 18-24 h.
b. After incubation, observe if the media change color and have any bubble in
the vial.
3. Result
E. coli
S. Typhoid
Glucose
⊕
+
Lactose
⊕
_
B. Metyl Red Test (MR) and Voges-Proskaurer Test(VP)
MR test and VP test used for the same general purpose: to distinguish between
E. coli and E. aerogenes and to lessen extent among other species as well.
1. Materials
Glucose-peptone water medium
Methyl red reagent
5% alpha naphthol, in absolute ethy1 alcohol
Slant cultures of E. coli and E. aerigenes
2. Procedure
a. Inoculate E. coli and E. aerogenes into two tubes of glucose peptone media
and incubate at 37℃ for 48 h.
b. MR test: add 5 drops of methy1 red reagent to one tube, records the result:
A red color means a positive test, showing that E. coli ferment glucose producing
enough acid to effect pH change from 6.0 to 4.4 (at pH 6.0) methyl red is red.
c. VP test: add 0.5ml of 5% alpha naphthol in absolute ethyl alcohol and
0.5ml of 40% potassium hydroxide into two tubes with E. coli and E. aerogenes,
respectively, stand for 15-30 min. A red color is developed (positive), which
indicate that the bacteria produce acelyl-methy1-carbiol by the fermentation of
glucose in the media.
3. Result
E. coli
E. aerogenes
MR test
+
─
VP test
─
+
C. Citrate Utilizing Test
Some bacteria can utilize citrate as their carbon source to provide the energy to
growth. The end production of this reaction is NH3, which makes the culture
medium’s pH>7. The indicator BTB (bromothymol blue) change color from green
to blue.
1. Materials
Slant cultures of E. coli and E. aerigenes
Citrate medium
2. Procedure
a. Inoculated E. coli and E. aerogenes into two tubes of citrate medium,
respectively, and incubate at 37℃ for 48 h.
b. Observe the color of the slant.
3. Result
Citrate Utilizing Test
E. coli
─
E. aerogenes
+
D. Indole test
Some bacteria produce tryptophanase, which can hydrolyze the tryptophan
into indole. Indole is colorless and invisible, but may reacts with dimethyl
aminobenzaluehyde to form rose indole.
1. Materials
Slant cultures of E. coli and E. Aerogenes
Peptone water medium
Kovacs reagent: para-dimethylaminobenzaldehyde dissolved in pure
amylalcohol and concentrated hydrochloric acid.
2. Procedure
a. Inoculate the E. coli and E. aerogenes into peptone water
media,
respectively.
b. Incubate at 37℃ for 18-24 h.
c. Add several drops of Kovacs reagent to the media and shake the culture
tubes.
d. A deep red color indicates a positive reaction.
3. Result
E. coli
E. aerogenes
Indole test
+
─
E. Hydrogen sulfide production test
Some bacteria possess the enzymes, which can split the sulfur containing
amino acids to produce H2S. The combination of H2S with lead acetate or ferrous
sulfate forms lead sulfide or ferrous sulfide, which is indicated by blackening the
medium.
1. Materials
Slant culture of E. coil and S. typhi.
Lead acetate agar or ferrous sulfide
Wire needle
Alcohol lamp
2. Procedure
a. Inoculate E. coli and S. typhi into the lead acetate agar with straight wire
needle.
b．Incubate at 37℃ for 24 h.
c. Positive test is indicated by the appearance of the black precipitates along
the stab track.
3. Result
H2S test
E. coli
─
F.
S. typhi
+
Pigments
Some bacteria can produce characteristically pigment, which give the
colonies or media a color.
1. Materials
24 hours broth cultures of S. aureus, S.epidermidis and P. aeruginsa
Agar slants or agar plates
wire loop
alcohol lamp
2. Procedure
a. Inoculate S. aureus, S. epidermidis and P. aeruginsa in the slants or plates,
respectively.
b. Incubate at 37℃ for 24-48 h.
3. Observe the color of colonies and media.
Experiment 5 Cultivation of Bacteria
It is possible that the most frequently used technique in the microbiology
involves the transfer of microbial growth from one environment to another. The
main purpose of inoculation is to get a pure culture of bacteria.
1. Materials
Agar plate, agar slant, semisolid agar, broth
Alcohol lamp
Agar slant cultures of S.epidermidis and E. coli
wire loop
A. Inoculating bacteria on the Petri dish
Many clinical specimens, such as feces, sputum and pus, etc, usually contain
various kinds of bacteria. The use of streak-plate method can often obtain discrete
colonies of the pathogenic bacteria and the pathogens can be isolated in pure
cultures. So the identification of the pathogens concerned becomes possible.
Procedure
1. Place the slant of the bacteria in left hand, remove the plug, flame the wire
loop and the mouth of the culture tube.
2. Insert the loop into the tube and pick the bacteria growth on the slant.
3. Remove the cover of the plate and hold the bottom in upright position with
left hand. Rub the inoculum on the top surface of the plate with the lip of the wire
loop, gently and continuously streak the inoculum back and forth until obtained the
streaking pattern you desired.
4. Replace the cover, flame-sterilize the wire loop and put it on the desk.
5. Label the plate, invert the Petri dish and incubate at 37℃ for 18-24 hours.
B. Inoculating bacteria on the slant
1. Flame-sterilize the wire needle.
2. Place a sterile agar slant in your left hand, remove the cotton plug, and pass
the mouth of the tube over the flame.
3. Touch the tip of wire needle very carefully to the surface of the bacterial
given in the slant.
4. Insert the wire needle containing a small amount of bacterial growth into
the slant culture tube.
5. Inoculate the bacteria on the surface of the slant by passing the tip of the
needle across the surface area in short swinging strokes, beginning near the end of
tube and coursing to the top of the slant, cover as much of the surface area as
possible.
6. Pass the mouth of the tube over the flame and replace the cotton plug,
flame-sterilize the wire needle.
7. Incubate at 37℃ for 18-24 h.
C. Inoculating bacteria on the semisolid agar
In addition to the direct microscopic examination of bacterial culture for
motility, another method in common used for determining this characteristic is the
inoculation and subsequent observation of the motility of bacteria in semisolid
media. Motility of bacteria is indicated by a diffuse growth of bacteria from the line
of inoculation.
1. Flame-sterilize the wire needle.
2. Place a semisolid agar tube in your left hand, remove the cotton plug and
pass the mouth of the tube over the flame.
3. Touch the tip of the wire needle to surface of the growth of bacteria and
pick a little.
4. Insert the wire needle into the semisolid agar straightly and return the
needle by the same way.
5. Pass the mouth of the tube over the flame and replace the cotton plug,
flame-sterilize the needle.
6. Incubate at 37℃ for 18-24 h.
Note:
1. Be careful to keep sterile technique to prevent contamination during the
laboratory periods. Do not speak and laugh.
2. Be careful not to cut the agar when streaking on the agar surface.
3. Do not pick up too much growth in inoculation, for there may be several
million organisms while your loop just touches to the growth. So, very small
(barely visible) quantities adhering to the wire loop are sufficient to affect a
successful transfer.
Experiment 6 Observation of Bacteria Growth
A. Observation of bacterial colony on the plate agar
Colony is made up of millions of bacterial cell derived from a single
bacterium. It is very regular (round) in shape and does not touch any others on the
plate agar. The colonies of different types of bacteria show different characteristics,
which can be used to identify the bacteria.
The characteristics of colony:
1. Size: small colony: 1mm;
medium colony: 2-3mm;
large colony: 3mm.
2. Form: circular or irregular.
3. Edge: entire or irregular.
4. Surface: convex, flat smooth, rough, dry or moist.
5. Transparency: transparent or opaque.
6. Color: some bacteria produce lipid-soluble pigments, which confined to the
colonies, others produce water-soluble pigments that diffuse into the medium.
B. Observation of bacterial growth in the broth
Bacterial growth in the broth may appear turbid, membrane-like growth or
sediment growth, according to the different types of bacteria.
C. Observation of bacterial growth in semisolid agar
Semi-solid agar is used to test the motility of bacteria. The growth of
non-motile bacteria is restricted to the inoculation line, and the medium keeps clean.
The motile bacteria spread throughout the whole medium while growing, so the
inoculation line is not visible and the medium appears turbid.
Experiment 7 Staining of Bacteria
Bacteria are small, almost colorless and difficult to observe even with the aid
of microscope. Fortunately, most bacteria readily react with various dyes, which
allow the morphology of bacteria cells to be distinguishing from extraneous
materials present in the stained smear. There are many methods to be used to stain
the bacteria, such as, simple stains, differential stains, special stains, and so on.
A. Gram's stain
The Gram's stain is a differential stain devised by Christian Gram in 1884.
This staining procedure is the most widely used which can differentiate the bacteria
into two groups, i. e. Gram-positive (G+), and Gram-negative (C-).
1. Materials
Slide
Staining rack
Wire loop
Normal saline
A set of Gram's stains (Gram's crystal violet, Gram's iodine, 95% alcohol, and
diluted Safranin solution)
Slant cultures of Escherichia coli and Staphylococuse epidermidis.
2. Procedure
2.1. Preparation of bacterial smear
a. Flame the wire loop to red-hot and cool it for a few seconds, do not touch it
to anything.
b. Place two small Drops of normal saline with the sterile loop on the slide.
c. Hold the culture tube with the left hand, flame the wire loop again.
d. Remove the plug from the culture tube with the right hand. Hold the plug
and do not let it touch anything else.
e. Pass the mouth of the culture tube through the flame, and remove a very
little bacterial growth from the agar slant with the sterilized loop. Do not
dig into the agar.
f. Pass the mouth of the culture tube through the flame again and replace the
plug.
g. Emulsify the growth with saline as figure and make a thin smear.
h. Do not forget to flame the wire loop again before lay it aside.
i. Allow the smear to dry in air or over flame.
j. Fix smear by passing the slide through the flame several times.
2.2 Gram's Stain
a. Cover the smear prepared on the slide with Gram's crystal Violet for l min
fist stain.
b. Wash with tap water and shake off excess.
c. Cover with Gram s iodine (mordant) for l min.
d. Wash with tap water and shake off excess.
e. Cover the smear with 95% ethyl alcohol (decolorize) for 30 seconds, wash
with tap water.
f. Cover the smear with safranin (counter-stain) for 1 min.
g. Wash with tap water and shake off excess.
h. Dry in air or with a fresh piece of hygroscopic paper.
i. Use the oil immersion lens to examine the smear.
3. Results:
Bacteria that retain the dye complex after washing and appear bluish-purple
are termed Gram-positive organism; those can not retain primary stain and appear
red after counter-staining are Gram-negative organism.
Note:
a. Decolorization is the most critical step in Gram’s stain. If you decolorize
too vigorously, all cells will lose their primary dye and appear red, on the other
hand, if you decolorize too gently, Gram negative bacteria will appear
bluish-purple.
b. Gram's stain is also affected by the incubating time of bacteria. It is
important for you to remember that Gram's stain, as a differential method, is valid
only with the cultures of 18-24 h.
Experiment 8 Distribution of Bacteria in the
Environment and Human Body
Bacteria distribute in every place of the environment including air, soil, water,
food, and the surface structures of human and animal body. Therefore, it is
important to understand the natural distribution of bacteria in order to establish the
aseptic concepts in medical practice and certain scientific experiments.
A. Bacteria in the air
1. Materials
Nutrient agar plate
2. Procedure
a. Put the agar plates at different places in the laboratory.
b. Remove the lid of the Petri dish.
c. Let the Petri dish to expose to the air for 10 min.
d. Replace the cover to the Petri dish, invert position, label and place in the
incubator for 18-24 h.
e. Count the number of colonies on the plate and observe their
characteristics.
B. Bacteria in running water and the pool water
1. Materials
Nutrient agar plate
Sterile cotton swabs
2. Procedure
a. Moisten a dry sterile cotton swab with water from tap water and pool water,
respectively.
b. Rub the surface of the agar plate with the cotton swab.
c. Replace the cover on the Petri dish, invert, and incubate at 37℃ for 18-24 h.
d. Count the number of colonies of bacteria on the plate and observe their
characteristics.
C. Bacteria on the finger tip
1. Materials:
Nutrient agar plate
Sterile cotton swabs
2. Procedure
a. Divide the plate with wax pencil into two equal squares.
b. Rub on the surface of the plate with the fingertip on one square before
disinfection. Disinfect the fingertip with 75% alcohol, and then rub on another
square.
c. Incubate at 37℃ for 18-24 h.
d. Count the number of colonies on the plate and observe their characteristics.
Observe the effect of disinfection with 75% alcohol.
D. Bacteria in the throat
1. Materials:
Blood agar plate
2. Procedure
a. Remove the lid on the blood agar plate.
b. Hold the plate at 30 cm from your mouth and cough violently to the surface
of the agar plate.
c. Replace the cover, invert and incubate at 37℃ for 18-24 h.
d. Count the number of the colonies and observe the characteristics.
E. Bacteria in the soil
1. Materials:
Chopped meat broth
Sterile pipet
Vaseline
2. Procedure
a. Add 0.5ml of 1/10 diluted soil (with water) to chopped meat broth by sterile
pipet.
b. Place the culture tube at 80℃ hot water for 30 min.
c. Add melting vaseline (about 1ml) to the surface of broth.
d. Incubate at 37℃ for 24-48 h.
e. Observe the color of chopped meat.
Experiment 9 Variation of Bacteria
Bacteria, like other living things, posses the traits of heredity and variation, i.e.
the resemblance and difference between parents and their progeny. In this
laboratory period, we shall observe the phenomenon of variation in some bacteria.
A. Smooth-rough variation
A change in the cell surface composition in certain species of bacteria may
occur, in which the bacteria may form colonies, which differ from those of the
original strain in the appearance. If the colony is mucous with glow glossy surface
on agar plate, we can call smooth colony; on the contrary, rough colony is dull and
matt. Usually, encapsulated bacteria form smooth colony, while non-encapsulated
cells produce rough colony. In general, the change in colony from smooth to rough
may involve some or all of followings:
1. The appearance of colony has altered.
2. The specific smooth-strain antigens are lost.
3. The susceptibility to certain bacteriophages has changed.
4. A reduction in virulence.
With varying the components of medium, the variation from S to R may be
reversible, i.e. from S to R, or vice versa.
B. H-O variation
Many strains of bacteria have flagella on the surface, The flagella may loss,
when the conditions which the bacteria need for their growth are changed, this
phenomena in termed the variation from H (Hauch ) to O (Ohne Hauch).
Proteus has peritrichous flagella distributed on the surface of bacterial body. The
bacteria spread rapidly over the plate when inoculation on the agar plate, we can
called swimming growth, but the swarming growth is inhibited if the bacteria
grown on the 0.1% phenylethy1 agar plate.
Experiment 10
Pathogenicity of Bacteria
The pathogenicity of bacteria includes invasiveness and toxin. Invasiveness is
a kind of capacity of bacteria to invade host tissues, which depends on the function
of enzymes and the formation of bacterial surface structure. There are many
enzymes produced by different bacteria species, for example, coagulase,
hyaluronidase, etc. The bacterial toxin are usually grouped as exotoxins and
endotoxins.
A. Hyaluronidase test
1. Materials
Rabbits
hyaluronidase
Needle
Alcohol
Indian ink Syringes
2. Procedure
a. Shave the hairs on the both sides of skin in the back of rabbit for
inoculation.
b. Sterilize skin with 75% alcohol.
c. Dilute the hyaluronidase with normal saline to 1:100 and mix equal volume
of Indian ink.
d. Inoculate intradermally the above mixture 0.5ml to one side of the back and
the mixture of ink and equal volume normal saline .S 0.5ml to the other side as the
control.
e. Examine the results 1 hour later, compare the diameter of
infiltration of ink on the both sides of rabbit.
B. Tetanus toxin test
1. Materials
Tetanus exotoxin
Mice
Syringe
Needle
2. Procedure
a. Inject intramuscularly the diluted Tetanus exotoxin 0.2 ml to one of the hind
legs of mouse.
b. Examine the results on next day. The inoculated leg shows spasimc paralysis
and the animal dies within 2/3 days.
C. Lecithinase test
1. Materials
C. perfringens
Fresh-egg-yolk plate
Anaerobic jar
Inoculating loop
2. Procedure
a. Inoculate C.erfringens on nutrient agar containing 10% sterilized
fresh-egg-yolk.
b. Put the plate in the anaerobic jar to culture anaerobic. Culture at 37℃ for
18-24 h.
c. Examine the result: the presence of turbid precipitation over the inoculated area
means positive. Which is due to the lecithinase, produced by C. Perfringens,
decomposes the lecithin of egg-yolk into glacerine and fatty acid, the latter
precipice on the inoculated area.
D. Plasma coagulase test
The production of coagulase is one of the major criteria employed to
differeciate S. aureus from S. epidermidis, because 90% S,aureus associated with
pathogenic processes. So, coagulase-positive Staphylococcus is considered as
pathogenic coccus.
1. Materials
The cultures of S. epidermidis and S. aureus.
Two tubes with 0.5ml rabbit plasma in it.
2. Procedure
a. Inoculate S. epidermidis and S. aureus into the rabbit plasma, respectively.
b. Incubate at 37℃ for 0.5h.
c. Examine the result: The rabbit plasma in which the S. aureus inoculated
coagulated, that means the coagulase-positive. The rabbit plasma in which
the S. epidermidis inoculated didn’t coagulated, that means the
coagulase-negative.
Experiment 11 Isolation and Identification of Pyogenic Cocci
Pyogenic cocci are most frequently encountered pathogenic bacteria. These
cocci commonly include four well-known genera, namely, Staphylococcus,
Streptococcus, Neisseria and Pneumococcus, which can be isolated from patients
with pyogenic cocci infection and healthy carries.
Although Gram stain of an isolate is helpful in identification of cocci, it is not
sufficient to differentiate pathogenic cocci from non-pathogenic. So the bacteria
must be isolated in pure culture and further identify according to antigenic structure
and biochemical test.
A technique commonly used in isolation of pathogenic cocci is the procurable
of microbial specimens on a swab, streaking the plate with swab, which is very
useful to get isolation of pure colonies.
1.
Materials
Blood plates, pus specimen, wire loop, alcohol lamp
2. Procedure
It needs 3-4 days to complete the procedure for isolation of the pathogen from
the pus specimen given.
Directly make smear and Gram's stain (Arrangement,
Pus swab
morphology, and stain characteristics)
Inoculate on blood plate 37C growth character
18h
A. first day
1. Streak with cotton swab on one edge of blood plate, with a back and forth
motion.
2. Cover the plate with cover and throw the swab into the cylinder with lysol
solution.
3. Sterilized the loop and drag it through the sector streaked with swab to pick
up bacteria deposited there.
4. Streak into the uninoculated area in a fashion similar to that performed in
the streak-plate technique.
5. Close the plate with the cover and inverted the plate.
6. Incubate the plate at 37℃ for 24 h.
B. Second day
1. Materials
Blood plate, rabbit plasma, slide, saline.
2. Procedure
a. Observation of the cultures: note colonial morphology and growth character
of bacteria. If the colonies are medium size, smooth, hemolytic and golden yellow,
Gram stain shows G+ and arranged in irregular clusters, the isolates may be S.
aureus. If the colonies are small in size, smooth, G+, and arranged in chains, the
isolate may be Streptococcus.
b. Plasma coagulase test
The production of coagulase is one of the major criteria employed to
difference S. aureus from S. epidermidis, because 90% S, aureus associated with
pathogen processes. So, coagulase-positive Staphylococcus is considered as
pathogenic coccus.
a. Place two loops of saline to the two area on the slide respectively.
b. Remove enough bacteria from a colony on the plate to prepare a heavy
suspension of bacteria.
c. Add one drop (or one loop) of rabbit plasma to the one of the suspensions,
quickly mix with the wire loop.
d. In few seconds, a positive result is visible clumping, and the control is still
homogenous liquid.
3. Sub-inoculate one colony of bacteria into fluid broth for antibiotics
sensitivity test.
C. Third day
1. Materials
Sterile cotton swab, blood plate, several paper discs containing different
antibiotics
2.
Procedure
a. Immerse the cotton swab with the broth cultures.
b. Spread with cotton swab over the surface of the blood plate.
c. Place the paper discs containing antibiotics on it and incubate the plate at
37℃ for 18 h.
d. Measure the inhibitory zone to determine the drug sensitivity of the
bacteria.
Experiment12
Effect of anti-microbial agent on bacteria
(Anti-microbial susceptibility tests)
Substances produced by one kind of microorganism can kill or inhibit others
are known as antibiotics or anti-microbial agents. There are many ways to
evaluating the effect of antibiotics on bacteria, including the dilution tests, such as
the broth tube and agar plate dilution procedures, and the disc agar diffusion test,
utilizing antibiotics impregnated discs. However, the most useful and practical
method for antibiotics susceptibility is the disc agar diffusion procedure. It is very
helpful for doctor to select the most effective drugs to render the infectious
diseases.
1. Materials
Nutrient agar plate
24 h broth culture of S. epidemidis
Sterile cotton swab
forceps
alcohol lamp
paper discs in filtrated with following;
penicillin
10 units
streptomycin
10 ug
gentamicin
10 ug
sulfadiazine
1 mg
tetracycline
30 ug
2. Procedure
a. Inoculate the S. epidermidis on the plate with sterile swab, make the
inoculation very heavy.
b. Remove antibiotics discs from vials and place on the surface of inoculated
agar plate with sterile forceps.
c. Invert the plate and incubate at 37℃ for 18-24 h.
d. Measure the diameters of inhibiting zones around the paper discs. The
susceptibility of bacteria is determined on the basis of Table 12-1.
Table 12-1 The susceptibility of bacteria to antibiotics
Antibiotics
penicillin
streptomycin
tetracycline
gentamicin
sulfadiaine
R:
resistant
MS:
moderately sensitive
HS:
highly sensitive
Inhibitory zone of
Antibiotics discs(mm)
10
10-20
20
10
10-14
15
10
10-14
15
10
10-18
19
10
10-15
15
Susceptibility
R
MS
HS
R
MS
HS
R
MS
HS
R
MS
HS
R
MS
HS
Experiment 13 Isolation and Identification of Enteropathogen Bacilli
A. Specimen collection
Stool should be fresh. Pick up the mucous or mucobloody portion of stool and
inoculated on the medium. If the stool can't be got, rectal swab can be used.
Besides, stool specimen should be immediately sent to the laboratory and
inoculated, or preserve in glycerol-saline.
A. Isolation and identification
It needs 3-4 days to complete the isolation procedure of enteropathogenic
bacilli from the stool specimen.
Stool swab
Direct inoculate
S-S agar plate
37℃, 24h
Pick up small, transparent
and colorless colonies
Double sugar iron agar
37℃,18-24 h
Gram's
stain
Semisolid agar
37℃, 24h
Observation of bacterial
biochemical
test
slide agglutination
test with known
antiserum
1. Materials: S-S agar plate, stool swabs, double sugar iron agar, semisolid agar
wire loop, alcohol lamp
3. Procedure
First day
a. Inoculate the stool specimen on the S-S agar plate with stool swab.
b. Incubate the plate at 37℃ for 24 h.
Second day: Observation of the growing character of bacteria and
sub-inoculation.
a. Observation of character of the bacteria on the S-S agar. S-S agar is a kind
of selective and differential medium, for it contains some substances, such as bile
salt, brilliant green, sodium thiosulfate, sodium citrate and so on, besides nutrients.
These substances can inhibit the growth of non-pathogenic enteric bacteria; salt can
promote the growth of Salmonella and Shigella, lactose and neutral red in the
medium act as indicators.
Generally, on the S-S agar, E.coli ferment lactose and produce acid, so the
colonies appear red color, and the enteropathogenic bacteria can't ferment lactose,
the colonies appear small, colorless.
b. Sub-inoculation
①. Pick up the small colorless colonies and inoculate into the double sugar
iron agar and semisolid medium.
②. Incubate the agar at 37℃ for 18-24 h.
Third day: Examination of the results and sub-inoculation
a. Observation of the results of double sugar iron and semisolid agar, Double
sugar iron agar contains lactose and glucose (lactose is much more than glucose)
and ferrous sulfate, sodium thiosulfate, phenol red acts as an indicator. When acid
i.e. pH<7 and gas, the medium shows yellow; alkaline pH>7, appear red. E.coli
ferment lactose and glucose and produce much acid. So the slant appears yellow
and there is gas in the culture. Some enteropathogenic bacteria ferment glucose but
not lactose, so the top of slant is still red (little acid has been volatilized), the
bottom of the slant appears yellow. If the bacteria produce hydrogen sulfide, the
slant appears black, because FeS is formed.
b. Slide agglutination test: with known antibody (such anti-salmonella or
anti-shigella serum), do slide agglutination test as usually fashion.
3. Sub-inoculation: pick up the bacteria from the surface of slant and inoculate
into sugar fermenting tube, and incubate at 37℃ for 18-24 h.
Fourth day:
Examination of results of sugar fermentation tubes, and final identification can be
determined.
Experiment 14 Acid-Fast Stain and Albert’s Stain
Acid-Fast Stain
Some bacteria, such as mycobacteria, do not readily stain under normal
staining procedures, but they can retain dyes with the aid of heating and resist
decolorization with diluted acid or acid alcohol. So these bacteria are termed
acid-fast bacteria.
1. Materials
Phenolized sputum sample from a patient
Carbol-fuchsion, acid alcohol, methylene blue
Staining rack
Slide
Wire loop
2. Procedure
a. Prepare the sputum smear, dry in air or over flame.
b. Cover the smear with carbol-fuchsion and gently steam over flame (do not
boil) for 3-5 min. Add the stain on the smear from time to time, do not allow the
smear to dry.
c. Allow the slide to cool, and wash with tap water.
d. Decolorize by dropping acid-alcohol on the smear until all the red color is
removed from the smear.
e. Wash with tap water.
f. Counter stain with methylene blue for l min.
g. Wash with tap water.
h. Allow the smear to dry and examine microscopically under oil immersion
lens.
3. Result
Acid-fact bacteria appear red;
Nonacid-fast bacteria appear blue.
Albert’s Stain
Some bacteria, such as Corynebacterium, have metachromatic granules in
their cell. These granules are composed of polymerized inorganic phosphates,
which represent a stored form of these elements widely used in various metabolic
processes of bacteria. The granules have a strong affinity for basic dyes. So, using a
basic dye, the granules are stained much more intensely than the remainder of the
bacterial cell.
1. Materials
Slant cultures of Corynebacterium diphtheria
Albert’s staining solution
Slide
Lugol’s iodine solution
Staining rack
Wire loop
2. Procedure
a. Prepare the smear of C.diphtheriticum.
b. Fix the smear with very gently heat.
c. Cover the smear with Albert’s staining solution for 5 min.
d. Pass the slide over flame back and forth, do not allow the stain to steam or
boil.
e. Drain the slide to remove excess dyes but do not wash with tap water.
f. Place Lugol’s iodine solution on the smear for l min.
g. Wash with tap water.
h. Allow drying and examining microscopically under oil immersion lens.
3. Results
Metachromatic granules appear very intense blue round dot within a pale
green stained cytoplasm.
Experiment 15 Toxigenic Properties of Corynebacterium Diphtheria (Elek
test)
Toxin production is one of the principal criteria for identifying C. Diphtherias
and for separating it from the non-pathogenic diphtheria. The test may be
performed in vivo using live animals or it may be performed in vitro (Elek test).
1. Materials
Filter strips containing diphtheria antitoxin (500 units), agar medium,
Toxigenic and non-toxigenic strains of C. Diphtheria.
2. Procedure
a. Using a sterile forceps, press a filter paper strip containing diphtheria
antitoxin below the surface of a molten but cooled agar medium in a Petri dish.
b. Allow the plate to dry at 35℃.
c. Streak a known non-toxigenic strain of C. diphtheria to the filter paper strip,
also streak a toxigenic strain perpendicular to the filter strip. As many as four or
five clinical isolates can be tested in this way on the same plate.
d. Incubating the plate at 35℃ and observe it after 1 to 3 days, the line of
growth will appear black or gray. Strains that are toxigenic will show a thin white
line of precipitate at an angle of 45 degrees from the culture streak. The white lines
are caused by the diffusion of the antitoxin and toxin in the agar medium; at the
points of optimal concentration they interact to produce a visible precipitate.
Experiment 16 Method of Virus Cultivation
Growth of viruses requires susceptible host cells capable of replicating the
virus. We are used to inoculate viruses with suitable cell cultures, animals, or
embryonated eggs. Prior to the inoculation of the specimen, it may be necessary to
eliminate bacteria from the specimen.
A. Cell and Tissue Culture Technique
Cell and tissue culture technique are the most widely used for the cultivation
of viruses, because this method is proved to be more sensitive, accurate and cheap.
The test conditions are easily managed, so the results are not affected by other
factors in the whole body (such as antibody complement, immune cells, etc). The
applications of cell and tissue culture method include: (1) routine isolation and
identification of viruses. (2) multiplication of viruses in established strains of
susceptible cells. (3) quantitative measurement of virus neutralizing antibodies. (4)
multiplication in tissue culture in sufficient quantity for the large-scale preparation
of virus vaccines. Cell and tissue cultures can be classified as following:
Primary cell monolayer
Monolayer cell culture
diploid cell strains
Continuous cell lines
organ culture
Example: The preparation of monolayer cell culture of human embryonic
kidney.
1. Materials
Fresh fetus (artificial abortion), sterile operating instrument, Hanks' solution,
0.25% trypsin solution, culture medium (Eagal, 199 or RPMI 1640 solution
containing 10% FCS and antibiotics),
2. Procedure
a. Sterilize the skin of the fetus with 2% iodine and take out the kidney.
b. Remove the capsule from the kidney, cut the surface area of kidney thus to
take off the cortex, discarding the medulla area, then place the cortex in a small
culture vial and chopped into small pieces(1-3mm), washed several times with
Hanks’ solution till the supernatant of the washing fluid to clear.
c. Digestion: put the chopped pieces into flask, add 10ml of 0.25% trypsin
solution with 0.1ml of antibiotics, adjust pH to 8.0 and digest in 37℃ water for 2 h
or icebox for 18 h, then washing three times with Hanks solution to remove the
remaining trypsin solution.
d. Centrifuge at 800-1000 rpm for 15 min, discard the supernatant, add 10ml
of culture medium and gently re-suspend the cells.
e. Dilute the cell suspension in 1:10 ratio and count the cells in
haemocytometer.
Cell number/ml=cell number in 4 big squares/4 ×100000
f. The cells suspension is transferred into culture medium at a concentration of
300000-500000 cells per ml.
Dispense into culture vials, 1ml being distributed into each vial. The vials are
stopped with rubber stoppers and incubated at 37℃. Monolayer cells can be
obtained in 3-7 days and could be used for the inoculation of viruses.
B. Embryonated Egg Technique
Embryonated eggs, which provide a sterile environment, are also used for the
multiplication of certain viruses.
1. Structure of the embryonated egg.
Immediately under the shell is the shell membrane, a tough fibrous material
which lines the entire shell but is readily separable from it; at the blunt end of the
shell membrane forms an air sac. The chorioallantois, a highly vascular membrane
serving as the respiratory organ of the embryo, lies directly under the shell
membrane throughout the entire egg. The chorioallantois is separated from the
amnion by the allantoic cavity, which contains 5-10 ml of fluid. The amniotic
membrane forms a sac, which encloses the embryo; the amniotic cavity within
which the embryo lies, contains approximately 1 ml of fluid, Term the amniotic
fluid. Attached to the emryo is the yolk sac, which contains nutrients for the
developing embryo,
2. Method of egg incubation (example by inoculation of allantoic cavity)
a. Clear fertile eggs and sterile the shell with 75% alcohol.
b. Place the eggs on the egg rack with the air sac up wards, incubation in a
humidified incubator at 38-39℃ for 10-12 days. After incubation for 3 days, turn
the eggs, 1-2 times everyday.
c. Candling eggs: discard infertile eggs, mark the position of the embryo and
air space on the shell with a pencil.
d. Inoculation of embryonated eggs, wipe surface with alcohol swab punch a
small hole on the shell, inoculated 0.2 ml of virus suspension, sealed the puncture.
Write your name and the name of the virus on the shell.
e. Incubated at 35-36℃ in an upright position, candling eggs everyday, after
72 h, take out and place them at 4℃ in a refrigerator overnight.
f. Harvesting of the allantic fluid: sterilize the upper part of the shell,
withdraw the fluid with a capillary pipet or syringe, about 6-8 ml allantoic fluid
may be obtained in one chick embryo.
g. Allantoic fluid should be placed in refrigerator until use.
3. Embryo inoculation technique.
a. Allantoic sac inoculation. 10-12 days embryos are used for production of
myxoviruses.
b. Amniotic sac inoculation: embryos 12-14 days old are used for primary
isolation of influenza virus. A small part of the amniotic chorioallantois membrane
is draw with a forceps so that the inoculum can be introduced directly into the
amniotic sac.
c. Yolk sac inoculation: embryos 6-8 days old are used for cultivation of
chlamydiae, rickettsiae and arboviruses. At this age the yolk sac almost fills the
entire egg. A hole is made in the air sac end. And the inoculum is introduced
directly into the yolk sac.
d. Chorioallantoic inoculation: embryos 11-13 days old are used for
identification poxviruses. An area free of vessels is selected by candling and the air
sac is located, and a artificial air sac is made by chipping the shell with a
sharp-pointed instrument over the marked area. The intact shell membrane is
exposed and slit by gently pressure. A second hole is made in the air sac end of the
egg. When gentle suction is applied through this opening, the intact chorioallantois
will drop away from the shell membrane beneath the first hole. The inoculum is
placed later into the chorioallantois.
Experiment 17 Serologic Tests of Viruses
The basic principle of serologic test of virus is identical to serologic reaction
of bacteria. We can apply it not only to detect unknown antigens with known
antibodies; here we mainly introduce the easiest and least expensive tests, i.e. the
non-serologic hemagglutination test and the serologic hemagglutination-inhibition
test.
A. Hemagglutination Test
Some viruses possess hemagglutinins (e.g. orthomyxoviruses and
paramyxoviruses), so the viruses may agglutinate erythrocytes. In vitro, it is called
hemagglutination. Hemagglutinated red blood cells show a diffuse blanket
covering the entire bottom of the well, whereas non-agglutinated cells, settle as a
small compact button on the Bottom of the well. Chick red blood cells are
generally the cells of choice for hemagglutination test of influenza viruses.
1. Materials
Allantoic fluid containing influenza virus, 0.5% chick erythrocyte, normal
saline, 1 ml pipet
2. Procedure
a. Set up 11 tubes on the rack, label the tube with pencil.
b. Add the materials into the tube as listed in the following table.
c. Shack the rack for a while and allow the tubes to stand in room temperature
for 45 min, don’t disturb them.
d. Observe and record the results as follow:
B.
-
no evidence of hemagglutination
++
50% hemagglutination
+++
75% hemagglutination
++++
100% hemagglutination
Hemagglutination-inhibition Test
Many viruses agglutinate erythrocytes, and this reaction may be specifically
inhibited by specific antiviral antibody. This principle is used to quantify the levels
of antibody, in order to diagnose the viral infection or to identify the serotype of the
virus. Diagnostic significance must be based on a rise in titer between acute and
convalescent sera, a 4-fold or greater titer increase is considered proof of active
virus infection. For most viruses, HI micromethod tests are now employed.
1. Materials
Allantoic fluid of influenza virus containing 4 HA unis/0.25ml, 0.5% chick red cell
suspension, inactivated patient's serum, normal saline, test tubes, rack, 1ml pipet
2. Procedure
a. Put 11 tubes in a row in the rack and mark each tube with pencil.
b. Add various materials into the tubes as listed in the following table.
c. Read hemagglutination degree of each tube as the method used in
hemagglutination test.
The highest dilution of serum to inhibit hemagglutination completely is the HI titer
of the serum (i. e. can inhibit 50% hemagglutination).
Table 20-1 Hemagglutination Test
Saline discard
1
2
3
0.9
0.25
0.1
0.25 0.25
4
5
6
7
0.25 0.25 0.25 0.25 0.25
8
unit: ml
9
10
0.25 0.25
11
0.25
0.5
viral suspension
0.25
0.25 0.25 0.25 0.25 0.25
saline
add 0.25 ml to each tube
0.5% chick RBC
Add 0.5 ml to each tube
Viral dilution
1/10
1/20
1/40 1/80
1/160
1/320
discard
1/640 1/1280
Shake and place the tube rack at room temperature for 45 min before reading
Results
0.25
the highest dilution of virus in the tube which shows”++”
hemagglutination degree is used to express 1 HA unit
1/2560
Control
Table 20-2 Hemagglutination -inbibition test
1
Saline
0.9
Patient serum
2
3
4
5
7
8
0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.1 0.25 0.25 0.25 0.25 0.25
Viral suspend
6
unit: ml
Contorl tubes
1
2
3
0.25 0.25 0.25(1:10)
0.25
-
-
-
sion(4u/0.25ml)
Add 0.25 to each tube
0.5% chick RBC
Add 0.5 to each tube
-
0.25
-
0.5 0.5 0.5
Serum dilution 1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280
Shake the rack vigorously and keep it stationary at room temperature
for 60 min before reading
Results
Table 20-2 Hemagglutination -inbibition test
Test tubes
unit: ml
Contorl tubes
1
2
3
4
5
6
7
8
1
2
3
Saline
0.9
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25 0.25
0.25
Patient serum
0.1
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25 -
-
Viralsuspension
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
-
0.25
-
0.5%chick RBC 0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Serum dilution
1/20
1/40
1/80
1/160
1/320
1/640
1/1280
(4u/0.25ml)
1/20
hake the rack vigorously and keep it stationary at room temperaturefor 60 min before reading
Results
Experiment 18
Pathogenic Fungi
The fungi, comprised of a large group of microorganism, are divided into two groups, single
cells and multiple cells; their nutrient requirement is not so high and usually grows on a simple
solid medium (Sabourand’s medium). They grow slowly and require a certain extent of moisture in
air; most of them require oxygen for growth. The optimal temperature for growth is about 22-28℃.
The pathogenic fungi can be identified by their characteristics of morphology and structure.
1. Materials
Prepared slides of Candida albicans,
Prepared slides of Dermatophytes.
Prepared slides of Crytococcus neoformans,
Sabourand’s agar slant cultures of various fungi
2. Procedure
a. Examine the prepared slides and observe the morphology and structure of fungi under the
light microscope.
b. Observe the cultures of fungi, notice the morphology of colonies.
c. Germ tube test: in the clinical laboratory one test used to identify C. albicans is the germ tube
test.
⑴. Remove a very small inoculum from the colony of C. albicans and transfer it to a small
glass tube containing 0.5ml of human or sheep serum.
⑵. Incubate the culture at 35℃ for up to 3 h. Observation can be made after 1 hour.
⑶. With a Pasteur pipet remove a drop of sediment from the serum tube and transfer to a slide
containing a drop of nigrosin.
⑷. Cover the lid with a cover slip and observe under high lens for the presence of small
pseudo hyphae (germ tube ) forming from the originally spherical cells.