1
INTRODUCTION
'ractical in Microbiology includes the
allowing exercises for undergraduate
.tudents.
l. Special staining
rwo smears are provided for special staining
[.e. one for Ziehl-Neelsen (ZN) staining and
:,ther for Albert's staining.
2. Identification of bacterial culture
Bacterial growth is provided for identification
on a culture plate as well as in liquid
medium.
3. Stoo]/Faeces examination
Faeces specimen is given for isolation and
identification of two pathogenic findings i.e.
ova and/ or cysts.
4. Spots
Five different spots in the form of specimens,
slides, glassware, media etc. are kept for
identification with relevant questions to
answer in one or two sentences.
How to deal with above exercises has been
described separately in subsequent chapters.
Each exercise is dealt in two parts:
1. The way student has to perform the
exercise and interpret.
2. The possible questions which may be
asked by the examiner and how to
answer these.
{Important Note: During microscopic
examination, students sometimes have a
problem in selecting power of objective lens
to be used for examination of different
3
exercises. To make it simple, hanging drClfJ
preparation and faeces should be examined
under high power (X40) objective. of
microscope while other stained preparations
(ZN staining, Albert's staining or Gram's
staining) are observed under oil immersion
objective (XlOO). Take care that condenser
of the microscope should be at the highest
level while using oil immersion objective.}
Certain precautions must be followed
while handling the live microorganisms in
Microbiology laboratory. These precautions
would help to minimise the transmission of
infective agents to laboratory personnel and
thus reduce the laboratory acquired health
hazards.
PRACTICAL INSTRUCTIONS TO
STUDENTS FOR WORKING IN THE
MICROBIOLOGY LABORATORY
1. Always wear a white coat or gown
while working in the laboratory. It will
prevent contamination of your clothes.
2. All material provided to you should
be regarded potentially dangerous and
capable of producing disease.
3. All cuts and burns, how so ever minor,
should be reported to your tutor or chief
laboratory technician.
4. Working bench/ table should be kept
free of books, papers and eatables.
5. Do not moisten the labels, pencils t
w1·th your tongue. e c.
6. Do not touch eyes, nose and m th .
ou with
your fin gers and hands, while .
in the laboratory. working
y Practical M
ICrot,,() C
4

13. Test tube mouth should be fl

7 In the event of accidental spilla~e of
. bacterial growth or other contaminated immediately before and after u s . an-ieo
111
of handling liquid culture. e cas('.
material, immediately report to your
batch tutor for disinfecting it thoroughly 14. Put used slides, coverslips and
0th
contaminated material in disinf er
before discarding. . ectanl
8. Always flame the bacteriological loop Jar. l

before and after use.
9. Culture tubes should be kept upright in
the racks and are not to be laid on the
bench.
10. Do not place cotton wool plugs on the
bench. Plugs should be held in hand
without touching the part that goes
inside the test tube.
11. While transferring culture material from
wire loop on to your slide, take care that
it should not fall off the wire loop.
12. If the wire loop contains infective
material, heat it slowly to avoid spurting.
15. Do not sit on the table tops.
16. Do not eat in the laboratory.
17. Students with long hair should tie thern
at the back properly to avoid risk of
contamination and catching fire.
18. Lenses of the microscope should be
cleaned with the lens paper or a clean
lint cloth. The oil immersion lens should
be cleaned with xylol. Material over the
stage and other parts of the microscope
should be wiped off.
19. Always wash hands with soap and wnter
after completing the laboratory work.

Essential Points to Remember

1. Follow the practical instructions while working in the microbiology laboratory.
2. ~b~erve st~ined preparations (ZN staining, Albert' s staining or Gram' s staining) under
011 unmers10n objective (XlOO).
3- Th~ c~ndenser of the microscope should be at the highest level while using oil immersion
ob1ective.
4. Examine
.
hancnng
o·
drop preparation
· and faeces under high
· power objective (X40) of the
m1eroscope.

- --
 2
COMMONLY USED METHODS

,n this chapter, staining 1nethods, Reagen ts

?reparation of hanging drop and stool/ A . Albert I or Albert stai11
:aeces exa1nination will be discussed. 1. Toludine blue .. 0.15 gn1
2. Malachite green .. 0.20 gm
I. STAINING METHODS 3. Glacial acetic acid 1 ml
:<or practical purposes, staining methods 4. Alcohol (95% ethanol) 2 ml
:an be conveniently divided into special 5. Distilled water to make .. 100 ml
,taining and Gran1 staining.
B. Albert II or Albert's iodine solution
4. Special Staining 1. Iodine 2gm
I . Albert's Staining 2. Potassium iodide 3 gm
,taining of Corynebacteria (Corynebacterium 3. Distilled water to make .. 300 ml
liphtherine and other corynebacteria) is done
JY this technique. 2. Ziehl-Neelsen (ZN) Staining or Acid-Fast
Staining
Vlct/10d Staining of Mycobacteria (usually
1. The smear is heated gently by flaming tubercle and lepra bacilli) is done by this
the slide fr01n underneath. It will fix the technique.
s1near. Do not overheat.
2. Cover the smear with Albert I (Albert's Method
stain) for 5 minutes. 1. The smear is heated gently by flaming
3. Drain off the whole stain without the slide from underneath. This will fix
washing. the smear.
4. Pour Albert II (iodine solution) over the 2. The carbol fuchsin stain is poured on the
smear so as to cover it completely, leave slide so as to cover the fixed sn1ear.
it for 2 minutes. 3. Gentle heat is applied to the underside
5. Drain off the Albert II solution without of the slide, by means of a spirit flame,
washing. until the stain just comn1ences to
6. Blot dry the smear with the help of filter steam.
paper. 4. The carbol fuchsin is left on the slide for
7. Examine this preparation under oil 5-10 minutes with intermittent heating
immersion (Xl00) objective. during that period. Care must be taken
to ensure that the stain does not dry out;
Important Note: Washing with water is to counteract drying n1ore solution of
wt done in this staining, only blot dry the stain is added to the slide and the slide
mear.} reheated. Heating of the stain is required

5

r 6
for penetration ot- the d ye into the cell
wall.
5. Wash with tap water. . .
6 The stained smear is decolounsed w~th
. 20% sulphuric acid and washed w1~h
water. This step should be repeated till
the pink/ red colour stops coming out:
{Note: In case of lepra bacilli 5% sulphuric
acid is used as M. leprae is less acid-fast.
Alternative method for decolourisation
is acid-alcohol (3 ml HCl and 97 ml
ethanol).}
7. The smear is counterstained with
methylene blue for 1-2 minutes.
Malachite green can also be used as
counterstain instead of methylene blue.
8. Wash with water and air dry.
9. Examine under oil immersion (Xl00)
objective.
Reagents
A. Zeihl-Neelsen's Carbo[ fuchsin
1. Basic fuchsin 1 gm
2. Phenol (crystalline) 5 gm
3. Alcohol (95% or absolute) .. 10 ml
4. Distilled water to make .. 100 ml
B. Methylene blue
1. Saturated solution of
methylene blue in alcohol .. 30 ml
2. KOH, 0.01 % in water .. 100 ml
8 . Gram Staining
Gram staining is done for identification of
bacterial culture provided to you. The smear
is prepared from culture plate provided to
you, it is first fixed and then stained.
Preparation of the Smear
1. Take a drop of water on a glass slide
with the help of wire loop.
2. With a straight wire or loop, touch the
bacterial colony and emulsify in drop of
Practical 1'1ir rq,
water to make a smooth suspc 11 .
is called smear. 10
~ 11. It
101
3. Let the smear be air dried. Don't
the smear for drying purposes. h~at
4. Dried smear is heated gently by flarn·
the slide from underneath. This is din~
to fix the smear. one
Method
1. Fixed smear is fully covered with crystal
violet solution (primary stain) for one
minute. (Other dyes such as gentian
violet or methyl violet may also be used
as primary stain.)
2. Pour Gram's iodine over the smear for
one minute.
3. Wash the smear with water.
4. Decolourise the smear with acetone
for 10-30 seconds taking care not
to overdecolourise. (Alcohol can be
substituted for acetone.)
5. Immediately wash with water to remove
the decolouriser.
6. Cover the smear with a dye safranin
(counterstain) for 1-2 minutes. (Dilute
carbol fuchsin or neutral red may also
be used as counterstain.)
7. Wash the smear with water.
8. The smear is then blot dried.
9. Examine under oil immersion (XlOO)
objective.
Reagents
A. Crystal violet solution
1. Crystal violet .. 0.5 gm
2. Distilled water to make 100 ml
B. Gram's iodine
1 gni
1. Iodine
2. Potassium iodide 2 gn1
100111!
3. Distilled water to make
C. A cetone
. Safrnnin and put it in the centre of a clean
1. Safranin .. 0.5 gm coverslip (Step 2) . . .
2. Distilled water to make .. 100 ml 3. Invert the glass slide contairung_ the
plasticine ring (Step 1) and place it on
the coverslip (Step 2) in such a manner
1. HANGING DROP PREPARATION so that the coverslip sticks with the
ranging drop is used to observe the motility plasticine and the drop remaining in the
f bacteria. It is prepared from the bacterial centre of the plasticine ring (Step 3).
rowth provided in liquid culture medium 4. Bring back the glass slide along ~ith
.1 a test tube. coverslip into the original position
(Step 4) .
lfetlwd (Fig. 2.1) 5. The drop now hangs beneath ~he
1. Make a ring of plasticine provided to coverslip, that is why it is called hanging
you and place it in the centre of a clean drop.
glass slide (Step 1). 6. Examine the preparation under the
2. Take a loopful of liquid bacterial culture microscope.

Step 1 - Glass
0
slir:;:i a plasticine ring

Step 2 - Coverslip with a drop of culture medium

Step 3 - Inverted glass slide from step 1 placed on to

coverslip in step 2

0
Step 4 - Glass slide in step 3 is turned upside down
Fig. 2.1 Hanging drop preparation
 8 Pract,cal ""
•· uc,-
0

Prt'cautitm~ under the high power ob'ecr

(i) Do not make the plasticine ring very and not under the low po: ive (X-ir,.
thick. or ,·ery thin. It should be of (XlO). er 0 bject1\ ~
appropriate thickness_- . (iii) Never examine the han .
. if . ging d
(ii) There is difficulty m . focussing ~he preparat 10n 1t has dried rop
hanging drop preparation when tluck preparation may show false r up. Dry
. . esu\~
plasticine ring is used. It nlight result motile bacteria may be seen 1.e
in breakage of coverslip while objective ·1
moti e. Th f . . as
ere ore, It IS always bett non.
. er to
lens is being lowered for focussing. repeat th e h angmg drop preparati
(iii) In case of thin plasticine ring, the for proper interpretation. on
hanging drop preparation touches the
glass slide and examination of drop
becon1es difficult. Ill. STOOL/FAECES EXAMINATION
Stool examination is done to find out 0,,,1
Examination (Fig. 2.2) and cysts of different parasites.
1. Always first focus the edge of a hanging
drop preparation under low power Metlzod (Fig. 2.3)
objective (XlO) of microscope. Wet preparations of stool specimen are
2. Turn the focus to high power objective prepared as follows:
(X-10) and obsenre for the motility of 1. Saline preparation as well as iodine
bacteria. Nlake sure that the edge of preparation of stool are made on the
the hanging drop should also be visible same glass slide.
under high power. 2. A drop of normal saline (0.9 per cent)
is put at one end of a clean glass slide
Precautions w hile on the other end of the same slide,
(i) Never use oil immersion objective a drop of iodine solution is put.
(XlOO) for hanging drop examination. 3. A minute portion of faeces is added
(ii) Always look for the motility of bacteria in both the drops with the help of a

Hanging drop preparation as visualised under Hanging drop examination as visualised under
X10 obj ective X40 objective
Ftg. _2. 2 Exam nat•on of hanging drop under the microscope
-------
 9

all wooden stick to make a .

pension. uniform Examination
1. Examine both the preparations (saline
.::overslip is gently pla d
. ce over each and iodine) under low power objective
Fens1on (saline and iodine) separately: (XlO) starting from one end to another in a
in~ care that no air bubble forms. ' particular given direction (Refer Fig. 2.4).
1m1ne the preparation under the 2. Any suspicious object, if found, should
::roscope (see below).
be studied under high power objective
(X40) for identification/confirmation of
the ova, trophozoites or cysts.
3. Always show your findings to the
examiner under high power objective
(X40).
•rmal saline Iodine
reparation preparation
Fig. 2.3 Stool preparation

tions Fig. 2.4 Stool examination: It should be

aeces in container should be mixed
roperly with wooden stick before
dding it either to saline or iodine
rops. Sometimes ova or cysts may
~ttle down in the container, mixing
nsures the uniform distribution of
1ese within the faeces.
f preparation has dried up, prepare
nother slide for examination purposes.
done in the direction of arrows starting
from one end
Precautions
(i) Never show your findings to the
examiner under low power (XlO) or
oil immersion lens (XlOO).
(ii) Do not report normal non-pathogenic
findings, e.g. cyst of Entamoeba coli.

Essential Points to Remember

. Understand and strictly follow all the working steps of various methods because these
will be used in different practical exercises later in subsequent chapters.
'. . In Albert's staining, smear is always blot dried and not washed with water.
,. Decolourisation step is very important in ZN staining (refer page 5).
.. Preparation of the smear is an important working step in Gram's staining (refer page 6).
1
_ Take all necessary precautions during preparation of the hanging drop and its examination
(refer page 7).
,. Follow proper precautions while making a wet preparation of the stool sample and
during its examination (refer page 9).
 CULTURE MED-..i
(

1
Two culture media most commonly used in B. Liquid Media
practical microbiology are solid and liquid 1. Peptone water (pH 7.4)
media. (i) Peptone
(ii) NaCl f, .
Solid media (iii) Water to make
lC.
1. Nutrient agar
2. Blood agar
3. MacConkey' s agar 2. Glucose broth

Liquid media
1. Peptone water (i) Nutrient broth <(:::;n•
2. Glucose broth extract - 1 ~()
(ii) Glucose (0.5%)
I. COMPOSITON OF MEDIA
A. Solid Media
II. IDENTIFICATION OF MEDIA

1. :~:~~::::roth <(::::ne extract- 1 %

(ii) Agar (2-3 %)
2. Blood agar
(i) Nutrient agar
(ii) Sheep blood (5-10%)
3. Ma cConkey's agar
(i) Peptone
(ii) Lactose
(iii) Sodium taurocholate
(iv) Agar
(v) Neutral red (indicator)
(vi) Water to make
A. Solid Media (Table 3.1)
Nutrient agar is semi-transparent ar :
pale, yellowish in colour (Fig. 3.1). It :~
not difficult to identify this medium. Bloo.1
agar is bright red in colour and it is opaqu~
(Fig. 3.2). Two layers are present in blooc
agar medium, one above the other. The lower
layer is of nutrient agar and upper layer IS
of sheep blood. In contrast, MacConkey's
agar is transparent and it is pink red lf\
colour (Fig. 3.3). If you keep your fin ger
outside below the culture plate and try
2.0 gm to see your finger through the medium,
1 .0 gm it is clearly visible; whereas the finger cannot
0.5 gm be seen clearly through the blood agar-
0.5 gm Absence of two layers in MacConkey' s agar
.. 0.075 gm is another differentiating feature. All the
100 ml three media are dispensed in a petridish.

10
 11
t.. til tu1 c Media

Table 3.1: Differentiating Features of Throe Solld Media

Character Nutrient agar Blood agar MacConkey's agar
l. Colour Pale yellowish Bright red (like blood) Pink red
2. Opacity Semi-trans parent Opaque Transparent
3. Two layers in the Absent Present Absent
medium

------·-
I
I

Fig. 3. 1 Nutrient agar Fig. 3.2 Blood agar

A
I.

I
J

Fig. 3.3 MacConkey's agar

 than glucose broth. Both the 11· .
while . d · qu1ct
are dispense 1n cotton plugo-ect .test Int,
t
J\vish b
U1'.\:
'

ir Glucose broth

ptone water and glucose broth

 In general, two smears are provided for
special staining, one for Albert's staining and
the other for Z iehl-N eelsen staining.
J. ALBERrs STAINING
A fixed bacterial smear is provided for
Albert's staining. In case of unfixed smear, fix
the smear by gently heating the slide from
underneath and then stain it.
1. Apply the method of Albert's staining
as described in chapter 2, page 5.
:. Examine the stained smear under
oil immersion objective (X100) of
rmcrnscope.
Report y our observations.
ssrvations
case of Corynebacterium diphtheriae,
~n coloured bacilli with bluish black
metachromatic granules are observed. B~cilli
are arranged in Chinese letter or cuneiform
arrangement (Fig. 4.1). Draw a well labelled
diagram of your observations using colour
pencils (take help of Fig. 4.1). Always focus
a good stained field of your smear because
the emphasis of this exercise is mainly on
the staining technique.
11. ZIEHL-NEELSEN (ZN) STAINING
OR ACID-FAST STAINING
A fixed smear is provided for Ziehl-Neelsen
staining. In case of unfixed smear, fix the
smear by gently heating the slide from
underneath and then stain it.
Procedure
1. Apply the method of Ziehl-Neelsen
(ZN) staining as described in Chapter
2, page 5.
2. Examine the stained smear under

A 8
r. 4, fCorynebacterium diphtheriae on Alb~rfs ~faining: _Green bacilli with metachromatic
granules {A) as seen under X100 magnificafton (8) diagrammatic representation

15
 Pus 0811 Q .4

Ans.

Acid-fast bacj
'Iii

Q.t
A B Am
Fig. 4.2 Mycobacterium tuberculosis on Ziehl-Nee/sen (ZN) staining: Acid-fast bacm·I (
(A) as seen under Xt 00 magnification (BJ diagrammatic representation AFB)
Q.
. .
oil 1mmers1on objective (X100) of tissue cells are stained blue (Fig. 4.2). Th An
rmcroscope. bright red bacilli are called acid-fast ba: Q
3. Report your observations. (AFB). D~aw a ~ell labelled diagram of your
A,
obs~rvations using colour pencils {take help
~ s of Fig. 4.2). Always focus a good stained field
In case of A Iucobacterium tuberculosis, bacilli of your smear because the emphasis of this
are 5een as bright red (acid-fast) while the exercise is mainly on the staining technique.
 I t.
r

Pus ce11

Acid-fast b
~

B
A
. 4 ., M cobacterium tuberculosis on Zie~/-Nee/se~ (ZN) stai:'ing: A cid -fas! bacilli (AFB)
Fig. - ~A) as seen under X100 magnification (BJ d1agrammat1c r:_epresentat,on

oil immersion objectiv e (XlOO) of tissue cells are _st~ined blue (Fig. 4.2). 'Int
microscope. bright red bac1lh are called acid-fast btl(
3. Report your observations. (AFB) . Draw a well labelled diagram of yo,
observations using colour pencils (take he\·
Observations of Fig. 4.2) . Always focus a good stained fiel
In case of Mycobacterium tuberculosis, bacilli of your smear because the emphasis of tht
are seen as bright red (acid-fast) while the exercise is mainly on the staining techniqut

Essential Points to Rem e n

1. Follow the working steps of staining of the smear Cl. -..:ctly (refer page 5).
2. Always examine the stained preparation under oil inlll"lersion objective (X100).
3 . Report a good stained field of the smear to the examine r.
4. Draw a well labelled coloured diagram of special staining.

Questions Related to
Albert's Staining
Glacial acetic acid
Q.1 Describe the method of Albert's staining. Alcohol (95 per cent ethanol)
Ans. Refer Chapter 2, Page 5. Distilled water • 11
Q.2 What are the constituents of Albert I and Albert II or Albert's iodine solutto
Albert II solutions? Iodine
Ans. The constituents of Albert I and Albert Potassium iodide
II are as follows: Distilled water
Albert I or Albert stain Q .3 What is Kl.ebs-Loejfler bacillus? ed as
Toludine blue Ans. The C. diphtheriae is also n aIIl fjrSI
Malachite green Klebs-Loeffler bacillus. It was
 nings

;cribed by Klebs, but was first Chinese letter

(ii) Pallisade
tivated by Loeffler. arrangement pattern
1at is the morphology of C.diphtheriae Q.1 1 What are the other. differen_ces between
Albert's staining? 7
C. diphtheriae and dzphthero~ds · .
ey are green bacilli with bluish Ans. C. diphtheriae Dzphtherozds
tck metachromatic granules. Bacilli (i) Grow on special Can gr?w
= arranged in Chinese letter or °
enriched culture on rd mary
neiform arrangement. media media
hat are metachromatic granules? (ii) Ferments glucose Ferments
,1ese are composed of poly- only and doesn't both glucose
,etaphosphates and represent energy ferment sucrose and sucrose
orage depots. (iii) Toxin is Non-toxic
/hat are other names for metachromatic produced
ranules? (iv) Pathogenic Commensal
'o lutin or Babes-Ernst granules. (Also refer Q. 10.)
Vhy are C. diphtheriae arranged m Q.12 Name some diphtheroids.
~hinese letter pattern ? Ans. (1) C. xerosis (found in the conjunctiva!
~hey are usually seen in angular sac)
:ashion resembling the letters V or (2) C. hofmanni (found in the throat)
L.. ( Chinese letter pattern) . This typical Q.13 Name some culture media usedfor growing
mangement is due to incomplete C. diphtheriae.
separation of daughter cells after Ans. (1) Loeffler' s serum slope (LSS)
binary fission. (2) Potassium tellurite blood agar
What is the morphology of C.diphtheriae medium
on Gram's staining? Q.14. What type of medium is Loeffler's serum
These are thin, slender, Gram positive slope?
bacilli (but tend to be decolourised Ans. Enriched medium
easily) and are club shaped due to the Q.15. What are the constituents of LSS
presence of metachromatic granules at medium?
one or both ends. Ans. The constituents of LSS medium are:
' How do diphtheroids differ from (i) Nutrient broth
C.diphtheriae on Albert's staining? (ii) Glucose
.. Diphtheroids are green coloured, (iii) Horse serum
short and thick bacilli. Metachromatic Q.16. Describe the morphology of C.diphtheriae
granules are absent. They are present colonies on LSS?
in pallisade arrangement and not in Ans. The colonies are small, circular, white or
Chinese letter pattern. creamy and glistening. These colonies
,O How do diphtheroids differ from appear after 6-8 hours of incubation.
C.diphtheriae on Gram's staining? Q.17. Nam_ e the selective medium used to grow
s. Diphtheroids C.diphtheriae C. dzphtheriae.
(i) Strongly Gram Weakly Gram Ans. Potassium tellurite blood agar.
positive, short positive and
Q.18. What . are the constituents of potassium
and thick thin bacilli tellunte blood agar?
bacilli Ans. It is blood agar contammg
. .
potassium
vcr 18
. h·•· 1·ts most
. . (O0-t o,,) which 111 ll1
Ans. Refer the Table below.
Practica1l"1
let,_

tellunte · . d thus acts as a Q.24 Wlzic/z biotype of C. diplitlie .

other t,actena an produce haemolysis on blood rinc 11,
selective ab-rent. . _ . fC di 11,theriae Ans. Mi tis biotype. ngnr) ~ 2/i ~ \
Q· ·19 I\-71,1f i::= the charactemflc ~ . . Id. ? Q.25 W/iat is the use of Hiss 's 5 t'
· f'[/unfe me mm . • ' er1.1n1 A11s. E.
co/0111/ on pota::=::=1u111 t . black Ans. Hiss s serum water is used f luQI,, :,)
A11::=. Colo~y of C. diph!heriae is grey or fermentation of various or tes~. el
coloured. . bl k • C. diphtheriae. sugar~. b
Q.20. I\!fry are colonies ~f C.diplztlreriae ac m
Q.26 What type of toxin is pr0d E
(ofour? . C.diphtheriae? !Iced Q 29 i,
,
A11• • It is due to reduction of potassmm Ans. I
Ans. Exotoxin
tellurite to tellurium. t
Q.21. Name o11e bacte~ium, other than Q.27 Describe some properties of
Q.30 l
produced by C. diphtheriae. exotoi
Corynebacteria, w/11clz pro_duces bla~k
coloured colouies on potassium tellurzte Ans. It is protein in nature. It consists f Ans.
ob
A d
fragments, an B. Both fragment .
111ediu111.
An:-. Staphylococcus maeus. required for toxi~ity. Fragment ; :
Q.22. Name three main biotypes of C.diphtheriae all the enzymatic activities wherea
rn tellulrife medium. fragment B is responsible for bind· .
.41;.- Gravis, intermedius and mitis. the toxin to the cells. It is extrem~; Q.3I
-.. ~3 F ":1 can three biotypes of C.diphtheriae potent toxin. It is converted into toxoi:
· Ans.
bt .' ..,~.,entiated? by heat or formalin.

Table (For Q.23): Three biotypes of C.diphtheriae compared

Q.3~
Character Gravis Intermedius Mitis
1. Morphology Short rods, few Long, poor Long rods, curved Ans
metachromatic granulation, barred prominent granules
granules, uniform forms with clubbed
stai::-1ing ends
2. Colony on tellu- Daisy head colony Frog's egg colony Poached egg colony
rite blood agar
3. Consistency of Brittle, not easily Soft, buttery, easily
Intermediate between
colonies emulsifiable gravis and mitis emulsifiable
4. Haemolysis Variable Non-hemolytic Usually haemolytic
5. Growth in broth Surface pellicle Q.
Turbidity in 24 hrs, Diffuse turbidity
clearing in 48 hrs,
with fine granular Ai
deposit
Biochemical tests
(i) Glucose Acid without gas
Acid without gas Acid without gas
(ii) Glycogen Acid without gas
(iii) Starch
Negative Negative
Acid without gas Negative Negative
Virulence
- Severe
- Moderate Mild
- ---

, the mode of action of C.diphtheriae
:n?
xin acts by inhibiting protein
~sis. It inhibits polypeptide chain
ation in the presence of NAD
activating the elongation factor
is "Park-Williams-8" strain?
the strain most commonly used
·)xin production.
does C. diphtheriae acquire the
:rty of toxin production?
toxigenicity of C. diphtheriae
!nds on the presence of a tox
!, of which beta phage is the most
ortant. Non-toxigenic strain may
rendered toxigenic by infecting
n with beta phage.
1t is lysogenic conversion?
! tox gene can be transferred from
! bacterium to another by lysogenic
:_teriophages. This process is known
lysogenic conversion.
w does concentration of iron influence
:in production of C.diphtheriae?
Lmg of iron per litre of culture
edium is optimum level for toxin
·oduction, while a concentration of
mg or more per litre inhibits the
,xin production. Reason for this
, not known. The repressor of tox
,ene appears to be an iron containing
irotein. When level of iron is more,
;uppressor is formed which inhibits
:oxin production.
\Jame the virulence tests used to
iemonstrate exotoxin of diphtheria
bacillus.
In-vivo tests
(i) Subcutaneous test } ~ a l .
..
(n) Intracutaneous test tests
moculation
In-vitro tests
(i) Elek' s gel precipitation test
(ii) Tissue culture test
I '1
Q.34 Describe the 'In-vivo' tests for
demonstratoin of exotoxin of C.diphtheriae
Ans. Two types of test are used viz.
subcutaneous and intracutaneous.
(i) Subcutaneous test
The growth from an overnight cu~t~re
on Loeffler' s serum slope is emulsified
in 2-Sml of broth and 0.8ml of this
emulsion is injected subcutaneously in
thigh of two guinea pigs, one ~f _wh!ch
has received an intramuscular mJection
of 500 units of diphtheria antitoxin
18-24 hrs. previously (this protected
animal acts as control). If the strain is
toxigenic, the unprotected animal will
die within 2 to 3 days with evidence of
haemorrhage in the adrenal glands.
(ii) Intracutaneous test
Two guinea pigs (or rabbits) are injected
intracutaneously with 0.1 ml emulsion
from growth on Loeffler' s serum
slope, one of these animals is protected
with 500 units antitoxin the previous
day (control) and the other is given
50 units of antitoxin intraperitoneally
four hours after the skin test, in
order to prevent death. If the strain is
toxigenic, the inflammatory reaction
at the site of injection, progresses to
necrosis in 48 to 72 hours in the test
animal but there is no change in the
control animal.
Q.35 What is the advantage of intra-
c~taneous ~est ave~ subcutaneous test fo r
demon stratzon of diphtheria toxin ?
Ans. Eigh~ to ten cultures can be te~ted on
a pa": of animals and the animals do
not die.
Q.36 Describe the 'In •t ,
. -vz ro tests for
demonstratzon of exotoxin of C diphth .
Ans. Thi
(i) Ele~'s gel precipitation .t est erzae.
S IS an i
A mmuno d"iffusion test
rectangular strip 0 f fil .
soaked in . . . ter paper
diphtheria antitoxin (1000