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1. Abbas Naseem (BDS)
abbas_naseem@yahoo.com
Dated: Nov 7, 2010

2. Chocolate agar
 Chocolate agar (CHOC) - is a non-selective, enriched growth medium.
 It is a variant of the blood agar plate.
 It contains red blood cells, which have been lysed by heating very slowly to 56 °C.
 Chocolate agar is used for growing fastidious (fussy) respiratory bacteria, such as
Haemophilus influenzae.
 These bacteria need growth factors, like NAD and hematin, which are inside
erythrocytes; thus, a prerequisite to growth is lysis of the red blood cells
2

3. Blood Agar
• A nutrient culture medium that is enriched with whole blood and used for the growth of certain
strains of bacteria.
• Contains mammalian blood (usually sheep or horse), typically at a concentration of 5–10%.
• BAP are an enriched, differential media used to isolate fastidious organisms and detect
hemolytic activity.
• β-hemolytic activity will show complete lysis of red blood cells surrounding colony,
• while α-hemolysis will only partially lyse hemoglobin and will appear green.
• γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity.
Different types of hemolysis on Blood Agar
3

4.  Cysteine Lactose Electrolyte Deficient agar
(CLED)
◦ CLED agar is used to isolate and differentiate urinary tract bacteria,
since it inhibits Proteus species swarming and can differentiate
between lactose fermenters and non-fermenters.
4

5. MacConkey agar (MAC)
 A selective and differential media used to differentiate between Gram
negative bacteria while inhibiting the growth of Gram positive bacteria.
 The addition of bile salts and crystal violet to the agar inhibits the growth
of most Gram positive bacteria, making MacConkey agar selective.
 Lactose and neutral red are added to differentiate the lactose
fermenters, which form pink colonies, from lactose nonfermenters that
form clear colonies.
Composition:
Bile salts
Crystal voilet
Lactose
Neutral red
• MacConkey agar is useful medium for cultivation of enterobacteria
• It contains a bile salt to inhibit non intestinal bacteria
• Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting
Salmonella and Dysentery group
5

6. Lactose fermenting
NON Lactose fermenting

7. Triple Sugar Iron medium
• Triple Sugar Iron medium is a differential medium that can distinguish between a
number of Gram-negative enteric bacteria based on their physiological ability (or
lack thereof) to:
• a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S.
• The medium contains 1.0% each of sucrose and lactose and 0.1% glucose.
• If only glucose is fermented, acid produced in the butt will turn it yellow, but
insufficient acid products are formed to affect the methyl red in the slant.
• However, if either sucrose or lactose are fermented, sufficient fermentation
products will be formed to turn both the butt and the slant yellow.
• If gas is formed during the fermentation, it will show in the butt either as bubbles
or as cracking of the agar.
• If no fermentation occurs (as for an obligate aerobe), the slant and butt will
remain red.
7

8. A 8-year-old boy developed sore throat and fever. On examination,
grayish exudates (pseudomembrane) is found over tonsils and
pharynx. The causative agent is grown on a medium which
produces blackish colonies as show in the photograph.
A. Identify the culture medium and the most likely the causative
organism.
B. Why the causative agent appears beaded on Albert‟s staining?
C. Enlist two complication of disease caused by this organism.
D. What is the primary mechanism in pathogenesis of Toxin mediated
complications.
8

9. A. Name the test, which is shown in the
photograph.
B. Why this test is performed?
C. Strip marked as “A” contains what
substance?
D. Identify the „Test‟, „Positive‟ and
„Negative‟ control and write the
appropriate alphabet in the answer for each.
9

10. Vibrio cholerae produces yellowish colonies
on agar as shown in the photograph.
A. Name the agar on which V. cholerae are
grown in the photograph.
B. This agar belongs to which class/type of
culture medium?
C. Comment on the motility of V. cholerae.
D. How would you differentiate V. cholerae
from members of enterobacteriaceae?
10

11. A. Name the given media.
B. Name the most likely causative agent in this patient?
C. Name the major constituents of this medium.
D. How much time is required for visible growth of this
organism on this medium?
 When grown on LJ medium, M. tuberculosis appears as brown, granule-like
colonies.
 The media must be incubated for a significant length of time, usually four
weeks, due to the slow doubling time of M. tuberculosis compared with other
bacteria (15-20 hours).
11

12. A pus swab received in the laboratory was inoculated
on blood agar, incubated for 24 hours and smear is
prepared from the colony of the bacteria and stained
with Gram stain.Examine the slide under the
microscope and
classify the organism on the basis of Gram staining.
Name the reagents required for Gram staining.
Write three conditions, which result in gram variability.
12

13. Sputum is received from a suspected case of pulmonary
tuberculosis and stained smear is provided.
A. Examine the slide and write your observation.
B. Which staining method has been opted for staining of
this organism?
C. Enumerate the reagents used in this staining method.
D. Name the mordant used in this staining method.
E. Name the organism, which require modified ZN staining.
• Requirements:
• Response sheet Pen / ball point Table
• Chair / Stool Binocular Microscope
• Z N stained slide positive for Mycobacterium tuberculosis.
13
Gold standart of T.B….to confirm it Z.N stain

14. In a remote village of Punjab, most of the population presented with
similar complains of sudden onset of vomiting and profuse diarrhea
with abdominal cramps. On examination, stool is like rice water in
appearance and contains mucous. Stool samples are collected in a
transport medium provided to you and to be sent to referral laboratory
for isolation of the causative organism.
Identify the transport medium provided.
Write the name of enrichment medium for stool samples.
Write two characteristic features of transport media.
Alkaline Peptone Water is an enrichment medium used for the cultivation of
Vibrio species from feces and other infected materials.
Peptones provide nitrogen, vitamins, minerals and amino acids essential for
growth.
Sodium chloride supplies essential electrolytes for transport and osmotic 14
balance and encourages the growth of Vibrio cholerae.

15.  Sabouraud agar is used to culture fungi and has a low
pH that inhibits the growth of most bacteria;
 also contains the antibiotic gentamicin to specifically
inhibit the growth of Gram-negative bacteria.
15

16.  Salmonella shigella agar plate with H2S
producing colonies
Black color colonies
16

17. Sensitivity plate
17

21. Urease Test

22. Anaerobic Culture Methods Anaerobic jar
• Anaerobic
jar
Figure 6.5

23. Bacterial Stains
Albert stains - reagents used to demonstrate volutin granules and cytoplasm in
diphtheria organisms.
Field's Stain - a rapid stain used commonly on thin films for malarial parasites.
Flagella Stain - allows for the direct observation of flagella
Gentian Violet Stain Recipe - Reagent used in the gram-stain procedure.

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Abbas Naseem
Dated: Nov 7, 2010