PRACTICAL

Medical Microbiology
PM 301-p
For
Third Year Pharmacy Students

By:
Prof. Dr. M.Seif Eldin Ashour

2006-2007

Acknowledgement

My deep thanks to Teaching Assistants at Faculty of Pharmacy

Modern Sciences & Arts University:

TA. Donia Abdel Rahman Mahmoud.

TA. Hebatallah Ezzat Abdel Raouf.
TA. Ahmed Farouk Mohamed.
TA. Mahmoud Abdel Khalek Hassan.
TA. Mai Mostafa Ahmed.
Deans Secretary. Heba Abdel Fattah

For their valuable and great effort in reviewing this book and
addition of colored figures.

Dr. M.S.E.Ashour

CONTENTS

I.

Page

BACTERIOLOGY
1. Biochemical reactions ..
2. Bacteriophage typing ..........
3. Staphylococci..
4. Streptococci and Pneumococci.................................................
5. Corynobacteria.......................................
6. Bacilli.......................................................................................
7. Clostridia.................................................................................
8. Neisseria...................................................................................
9. Enterobacteriaceae (Lactose fermenters)
10. Enterobacteriaceae (Non Lactose fermenters)........................
11. Pseudomonas...........................................................................
12. Brucella.....................................................................................
13. Vibrios.......................................................................................
14. Mycobacterium..
15. Spirochaetes..

1
7
8
12
18
21
24
28
31
37
44
47
49
52
55

MYCOLOGY:
1. Superfacial mycosis...................................................................
2. Systemic mycosis.......................................................................

II.

57
61

IMMUNOLOGY:
1. Agglutination reactions...............................................................
2. Precipitation reactions................................................................
3. Toxin- antitoxin neutralization....................................................
4. Complement fixation test..
5. Fluorescent antibody techniques...............................................
6. Radioimmunoassay (RIA).
7. Enzyme linked immunosorbent assay (ELISA).................

66
70
72
74
76
78
79

15 week plan
1st week

Biochemical reactions and Bacteriophage typing

2ndweek

Staphylococci, Streptococci and Pneumococci

3rd week

Corynobacteria and Bacilli

4th week

Clostridia and Neisseria

5th week

Enterobacteriaceae (Lactose fermenters)

6th week

Enterobacteriaceae (Non Lactose fermenters)

7th week

Pseudomonas, Brucella and Vibrios

8th week

Mid term exam

9th week

Mycobacterium and Spirochaetes

10th week

Mycology

11th week

Agglutination reactions

12th week

Precipitation reactions and Toxin- antitoxin neutralization

13th week

Complement fixation test, Fluorescent antibody techniques,

Radioimmunoassay (RIA), Enzyme linked immunosorbent assay (ELISA)

14th week

Rivision

15th week

Final exam

BIOCHEMICAL REACTIONS
Bacteria are identified by biochemical reactions which are based on
difference among bacterial species in their h1etabolic activities and
enzymatic capabilities. Diagnostic tables showing biochemical reactions are
constructed for identification of many genera and species of bacteria.
To be valid for identification of bacteria the tests should be
standardized and should be done on bacteria in pure culture.
Examples of biochemical
identification of bacteria are:

reactions

in

common

use

for

1. Sugar fermentation: Bacteria vary as regards their fermentative

capabilities of different sugars, with acid production only or with acid and
gas production. Sugar media used are composed of 1%of the test sugar in
peptone water and Andrad's indicator which turns dark pink if acid is
produced. For detection of gas production a small inverted tube (Durham's
tube) is placed in each culture tube in which gas bubble can be
demonstrated. The sugars commonly tested for are: glucose, lactose,
maltose, mannite, sucrose and salicin.

2. Methyl red (M.R.) test: is used to detect the production of sufficient acid
during glucose fermentation to reduce the pH of medium to a level below
4.5 leading to change of the colour of the indicator to red (e.g. E. coli is
M.R. positive). The organism is grown on glucose phosphate for 48 hr. and
few drops of methyl red are added.

3. Voges-Proskauer (V.P.) test: Some bacteria ferment glucose with the

production of acetyl methyl carbinol. This substance can be tested for by the
addition of concentrated KOH to the organism growing on glucose
phosphate peptone medium for 48 hr. This gives pink colour in a positive
test. An organism of the Enterobacteriaceae family is usually either M.R.
positive and VP negative or MR negative and V.P. positive.

4. Indole test: Certain bacteria are able to decompose the amino acid
tryptophane present in peptone to indole. This is then tested for by a
colourimetric reaction. Using kovacs or Erlichs reagents containing Paminobenzaldehyde which gives a red coloured compound. The organism is
inoculated in peptone water and after incubation at 37C for 24 hrs, the
reagent is added.

5. Hydrogen sulphide (H2S) production: Some bacteria decompose

Sulphur containing amino acids to form H2S. This is detected using lead
acetate paper strip which turns black due to the formation of black insoluble
lead sulphide. The organism is grown in peptone water with a suspended
lead acetate paper strip.

6. Catalase test: Some bacteria (e.g. staphylococci ) are able to produce

catalase enzyme which causes the release of O2 bubbles from hydrogen
peroxide when it comes in contact with it.

7. oxidase test: Cultures of oxidase producer bacteria (e.g. Neisseria) if

treated with few drops of oxidase reagent (1% solution of tetramethyl-pphenylene diamine dihydrochloride)show the rapid development of deep
purple colour. This test is done by picking up a portion of the suspected
colony to be tested and is streaked on a strip of filter paper impregnated
with 1% of freshly prepared oxidase reagent.

8. Urease test: Urease producer bacteria can split urea with the release of
ammonia. Such bacteria (e.g. proteus) if grown on a medium containing urea
and phenol red indicator, the medium turns red due to the release of
ammonia.

9. Coagulase test: Staphylococcus aureus produces coagulase enzyme

which causes coagulation of plasma by converting fibrinogen to fibrin.
This could be tested for on slides (bound coagulase) or in a test tube (free
coagulase).

Commercial API kit systems: are available for biochemical identification

of some bacteria, e.g. Enterobacteriaceae, Streptococci ... etc. It is composed
of a plastic strip with miniaturized cupules containing dehydrated media and
reagents. Each cupule has a small hole at the tip in which is dropped a saline
suspension of the isolated organism. The strip is then covered and incubated
at 37C for 24-48 hrs in the presence of humidity. The biochemical profiles
are determined and interpreted according to provided charts

API 20E Test System.

Identify and color the illustrative diagrams of the demonstrated
biochemical reactions.
1. Sugars fermentation:
The routinely used sugars are:
glucose, lactose, maltose, mannite, sucrose, salicin.

BACTERIOPHAGE TYPING
Bacteriophages are viruses that infect bacteria and cause their lysis. A
given phage type infects a limited range of susceptible bacteria. Some
phages have a wide bacterial host range. Some phages attack only one or
few closely related strains of bacteria belonging to the same species.
Tracing of the source of infection in an outbreak of hospital wound
sepsis or of food poisoning is very important. This can be achieved by phage
typing of both the bacteria isolated from clinical specimens obtained from
the patients and of the bacteria isolated from the attendants or suspected
sources.
The test is done by inoculating a plate of a suitable culture medium
with the strain to be tested for its phage type. The plate is dried. A drop of
each of the recommended set of different phages is put on different areas of
the plate. The plate is incubated at 30C for 24 hours. The plate is to be
examined for areas of lysis of the bacterial growth due to certain phages.
This determines the phage type of the strain.

The phage type of the staphylococcal strain tested is

52, 52a, 80

1) Staphylocoocus
I) Classification according to endopigment production:
1. Staphylococcus aureus

golden yellow pigment.

2. Staphylococcus epidermidis
(Staphylococcus albus)

white pigment.

3. Staphylococcus saprophyticus

lemon yellow pigment.

II) Morphology and staining reaction:

Gram-positive cocci which are arranged in grape-like clusters.

III) Culture characteristics:

Grow well on ordinary media aerobically and, although less well,
anaerobically; optimal temperature 37C.
Colonial appearance:
Staph. aureus: typically, golden colonies but pigmentation varies from
orange to white.
Staph. epidermidis: white colonies.
Pigment production by Staph. aureus is often poor after 24 h incubation at
37C on nutrient agar or blood agar; this important differential feature can
be enhanced by:
1- Leaving the plates in the light at room temperature.
2-Pigmentation is encouraged by growth on special media, e.g. milk agar.
Selective media:
Staphylococci will tolerate sodium chloride in concentrations of 5-10%.
Salt-containing agar and broth are useful in isolating staphylococci from
samples likely to contain large numbers of other bacteria.

IV) Diseases caused by Staph. aureus:

Staph. aureus, the main pathogenic species, is an important pyogenic
organism. Below is a list of some of the diseases it causes.
1. Superficial infections: pustules, boils, abscesses
2. Deep infections: septicaemia, endocarditis, pyemia, osteomyelitis,
pneumonia.
3. Toxic food poisoning.
4. Toxic shock syndrome.
5. Skin exfoliation: toxic epidermal necrolysis (Ritter-Lyells disease).
Staph. epidermidis, is of much lower pathogenicity and rarely causes
infections in healthy people. However, it has emerged in recent years as
an important pathogen of implanted metal and plastic devices.
Staph. saprophyticus, is an important cause of urinary tract infection in
sexually active women.

VI) Illustrative pictures for the M.O. :

1. Microscopical Examination:

2. Culture characteristics:
a) Nutrient agar

Golden yellow colonies

Lemon yellow colonies

White colonies

....

b) Mannitol salt agar (MSA):

3. Biochemical reactions:
a) Coagulase production test (tube Coagulase test):
5 drops of overnight broth culture of the organism + 0.5 ml human or
rabbite citerated plasma incubate at 37C for 6-12 hours
coagulation of the plasma

b) Catalase test:

10

c) Phosphatase test:
Principle:
M.O. release phosphatase which break phosphate moiety attached to the
organic compound to utilize phosphate.
Procedures:
Phosphatase

N. agar contains ph.ph. diphosphate

Pink color

free ph.ph.

4. Phage typing:
1. Draw the provided plate of phage typing of Staph. aureus strain.
2. What is the phage type of the provided staphylococcus strain?
3. What is the value of phage typing?

11

NH3

2) Streptococcus
I. Classification:
Complete

Colourless, clear,
sharply defined
zone

Pyogenic
streptococci

Partial

Greenish
discoloration

Viridans
streptococci and
pneumococci

None

No change

Enterococci

II. Morphology and staining reactions:

Gram-positive spherical or oval cocci in pairs or chains.
III. Culture characteristics:
Enriched media grows well on blood agar.
Selective media containing an amino glycoside antibiotic or 1:500,000
crystal violet inhibit other bacteria in a mixed culture but permit growth of
streptococci.
Aerobic bacteria but grow well anaerobically. Growth enhanced by 10%
carbon dioxide.

IV. Diseases produced by Streptococcus pyogenes:

*
*
*
*
*
*
*

Tonsillitis and pharyngitis.

Peritonsillar abscess (quinsy).
Scarlet fever.
Otitis media.
Mastoiditis and sinusitis.
Wound infection; may lead to cellulitis and lymphangitis.
Impetigo. (Skin infection causes yellowish discoloration)
12

*
*

Erysipelas; an acute lymphangitis of the skin.

Puerperal sepsis.

Post-streptococcal diseases:
*
*
*

Rheumatic fever.
Glomerulonephritis.
Erythema nodosum.

VI. Illustrative pictures for the M.O.

1. Microscopical examination.
a) Streptococci (in smear)

b) Streptococci pneumonia (in tissues)

13

2. Culture characterstics:
a) Blood agar plate showing:

- haemolysis: ................
-haemolysis: ..........
-(no haemolysis): ...............

3. Biochemical reactions:
a) Bacitracin sensitive strain:
(Strept. .)

14

b) Optochin sensitive strain:

(Strept. )

Strept.
Viridan

Streptococcus
Pneumoniae
c) Bile solubility test:
Principle:
Reading of the test:

15

d) Inulin fermentation test:

5. ASLO test:

Procedure:
1. Serial dilution of the patient's serum (1/100, 1/200, 1/400, 1/800, 1/1600,
1/3200 ... etc) is done.
2. Equal amounts of streptolysin O toxin is added to each tube.
3. The mixture is incubated at 37C for 10 min.
4. Human (or rabbit) group O 5% RBC is added to each tube.
5. Tubes are shaken and incubated at 37C for 30 min.
6. Read the titre of ASLO as the highest dilution of use serum which shows
no haemolysis.
7. Two controls are included:
A serum control: containing no toxin no haemolysis.

16

 A streptolysin toxin control: containing no serum haemolysis.

The titre is expressed in Todd's units. A titre of 200 Todd units or more is
significant.
This test is a toxin antitoxin neutralization test.
Read the titre of the demonstrated ASLO test.

VII. Tabulate the differences between Strept. pneumoniae and the viridans
streptococci:

17

3) Corynebacterium
I.

Classification:

Corynebacterium
diphtheriae
Gravies strains

Intermedius strains

Mitis strains

appearance in film
from Loeffers
medium
Club-shaped, few
metachromatic
granules
Short irregularly
staining rods without
metachromatic
granules but in
Chinese character
arrangement
Classic morphology
with numerous
granules and typical
arrangement

Colonial type on
tellurite medium
Flat, grey with raised
center and irregular
edge; radial striations
develop to form a
daisy-head
Small, smooth colonies
of uniform size; greyblack with paler
periphery
Medium-sized,
circular convex,
glistening and black

II. Morphology and staining reaction of C. diphtheriae:

Pleomorphic Gram-positive rods or clubs which divide by snapping
fission, so that adjacent cells lie at different angles to each other forming V,
L and W shapes - So-called Chinese-character arrangement
III. Culture media:
An aerobe and facultative anaerobe; optimum temperature at 37C. Does not
grow well on ordinary agar, and media containing blood or serum are
required.
Selective media are necessary for isolation from clinical specimens.

18

Selective media:
1-Loeffers serum medium:
C. diphtheriae grows rapidly, faster than other upper respiratory tract
bacteria present in clinical material: the morphology develops particularly
well and smears made as soon as
8 h after inoculation may show a typical appearance.
2-Blood tellurite agar
After 48 h incubation, corynebacteria produce characteristic grey-black
colonies due to their ability to reduce potassium tellurite to tellurium.
IV. Laboratory diagnosis of a case of diphtheria:
1. Specimen
2. Film
3. Culture
Laboratory diagnosis of a carrier for diphtheria:
1. Specimen
2. Culture
3. Virulence tests

a- Guinea pig inoculation: inject suspension of the isolated

strain of C. diphtheriae into two guinea pigs, one protected
with diphtheria antitoxin.
Result

Unprotected animal

Toxigenic strain
Non-toxigenic strain

Death in 2-3 days

Survival

Antitoxin protected
animal
Survival
Survival

2. Gel-precipitation (Eleks) test: a filter paper strip previously

immersed in diphtheria antitoxin is incorporated into serum agar
before it has set; the strain of C. diphtheriae under investigation

19

is then streaked into the agar at right angles to the filter paper
strip. Incubate at 37C.
Observe: after 24 h and 48 h for lines of precipitation
indicating toxin-antitoxin interaction.

V. Illustrative pictures for the M.O.

1. Microscopical examination:
a) C. diphtheriae (Gram stain)

20

2. Culture characteristics:
a) Blood tellurite

b) Lofflers serum

21

4)Bacillus
I. Classification:
Pathogenic:B. anthracis causes anthrax
Non Pathogenic:(Saprophytic but may cause disease):B. cereus
B. pumilis
B. stearothermophilus
B. subtilis
II. Morphology and staining reaction:
Morphology: (B. anthracis )
Gram +ve large rectangular bacilli usually arranged in chains
Non motile.
Sporulated in vitro (oval and central spores stained by spore stain).
Capsulated in vivo (consists of polypeptide-D-glutamic acid).
N.B:- Other Bacillus sp. are motile and non capsulated
III. Culture characteristics:
Facultative anaerobe; grows readily on ordinary media over a wide
temperature range (optimum 35C). Best temperature for sporulation is
lower, 25-30C.
Colonies are large, dense, grey-white, matt and irregular so-called medusa
head of curled hair lock appearance.
Blood agar: there is only slight haemolysis around the colony, a differential
feature because other Bacillus species are markedly hemolytic.
Broth cultures: develop a thick pellicle.

22

Gelatin stab cultures: inverted fir tree; liquefaction is late, starting at the
surface.
IV. Diseases in man and clinical presentations:
B. anthracis causes anthrax in man and animals.
In man infection occur from infected animal and may be:
1) Cutaneous anthrax: infection occur through damaged skin or mucous
membrane.
2) Pneumonic anthrax: infection occurs by inhalation of spores.
3) Intestinal anthrax: infection occur by eating infected meat
V. Clinical specimens:
Cutaneous anthrax
Pneumonic anthrax
Intestinal anthrax

skin swab
sputum
stool

VI. Illustrative pictures for the M.O.:

1. Microscopical examination:
a) B. anthracis (Gram stain)

23

b) B. anthracis (spore stain)

2. Biochemical reactions:
a) Starch hydrolysis:

b) Casein hydrolysis:

24

c) Gelatin hydrolysis:

25

5)Clostridium
I. Species of medical importance and diseases in man:

Disease
Cl. tetani

Tetanus

Cl: perfringens
Cl. septicum
Cl. novyii

Gas gangrene

Cl. histolyticum
Cl. sporogen
Cl. botulinum

Botulism

Cl. difficile
colitis

Antibiotic-associated

1. Microscopical examination:
Large (3-8 0.5 mm) rods, sometimes pleomorphic, filamentous forms are
common: Gram-positive but may stain irregularly or may be Gram-negative
in older cultures. Spores: all species form endospores which are usually
bulging, i.e. wider than the bacterial body; sometimes useful in
identification, e.g. Cl. tetani.
Note that Cl. perfringens (the most common human pathogen) forms spores
with difficulty. Motile, with peritrichous flagella (Cl. perfringens is nonmotile).Capsule: Cl. perfringens has a capsule, but most are non-capsulated.

26

II. Illustrative pictures for the M.O.

1. Microscopical examination:
A) Cl. tetani (Gram stain):

B) Clostridum spores:

2. Culture characteristics:
a) Anaerobic jar (gas pack):
A- This is a jar with air-tight cover made of transparent plastic.
B- A cold catalyst made of aluminum pellets coated by palladium and
covered by a gauze and fixed to the under surface of the lid. The organism
is inoculated on plate of blood agar and placed in the jar. Open the envelop
and put 10 ml. water in it, then put the envelop inside the jar. Be sure that
27

the catalyst and the indicator are in their place fixed to the under-surface of
the cover. Then cover the jar by the air-tight cover. CO2and H2 will evolve
from the envelop. The catalyst will facilitate union of H2 with the O2 inside
the jar making water drops. So the condition will become anaerobic within
short time and the indicator will loose the Color.

B) Robertsons cooked meat media:

This is broth containing cooked minced meat. This meat act as a source of
perioxidase and catalase enzymes that is deficient in anaerobes making them
able to grow in this media.

28

3. Biochemical reactions:
a) Litmus milk (Stormy clot):
Saccharolytic action of clostridia produce acid and large amount of gas when
cultivated on litmus milk medium

29

6) Neisseria
I. Classification:
i. Pathogenic Neisseria:
a) N. gonorrhoeae, or gonococcus:
Enters through mucous membrane of genito-urinary tract or conjunctiva,
cause gonorrhoea and some cases of purulent ophthalmia. Usually seen in
direct smears of pus from urethritis and cervicitis.
B) N. meningitidis, or meningococcus:
Enters through mucous membrane of upper respiratory tract, to blood stream
and central nervous system.
Healthy nasopharyngeal carriers provide the reservoirs of infection, cause of
cerebrospinal fever. Also, it is called meningococcal meningitis, found in
cerebrospinal fluid in cases of meningitis and usually easily seen in direct
smears.
ii. Harmless Neisseria:
N. catarrhalis, N. pharyngeus, are commensals of the respiratory tract.

II. Morphology and staining reaction:

Neisseria are Gram-negative cocci kidney shaped arranged in pairs with the
adjacent surfaces flattened or slightly concave.

III. Culture characteristics:

Media used are chocolate agar or Thayer-Martin agar. Incubation is done at
37C, in an atmosphere of 3-7% CO2.

30

IV. Diseases and clinical specimens:

A- N. gonorrhea causes 2 types of infections:
1. Venereal infection (gonorrhea):
2. Non-venereal gonococcal infections:
a. Opthalmia neonatorum
b. Vulvovaginitis
c. Gonococcal bacteraemia
A) Diagnosis of acute gonorrhea in males:
1. Specimen: Urethral discharge.
2. Direct smears stained with Gram-stain show Gram-negative diplococci
intra- and extracellular, i.e. inside and outside pus cells.
B) Diagnosis of other gonococcal infections:
1. Specimens:
The morning drop in case of chronic gonorrhea in males.
Urethral and cervical discharge in case of acute or chronic gonorrhea in
females.
Conjunctival discharge in case of ophthalmia neonatorum.
Synovial fluid in arthritis or blood sample in endocarditis.
2. Direct smears stained with Gram: It is hard to detect the organism either
due to the presence of normal flora or due to the low number.
3. Culture: Media used are chocolate agar or Thayer-Martin agar. Incubation
is done at 37C, in an atmosphere of 3-7% CO2.
B- Meningococci cause meningitis which tends to occur in epidemic form,
i.e. epidemic cerebrospinal meningitis.
Laboratory diagnosis of meningococcal meningitis
Specimen: Cerebrospinal fluid (CSF), blood and nasopharyngeal swab.
1. CSF: It is obtained by lumbar puncture and examined in the following lines:
a. Physical inspection: It is turbid due to large number of pus cells.
31

b. Cell count: Total number of cells is 20,000/cm.

c. Direct smears from the deposit of centrifuged CSF are stained with Gram
stain, these show Gram negative cocci arranged in pairs both intra-and
extracellular.
d. Culture: Media used are chocolate agar or Thayer-Martin agar. Incubation
is done at 37C in an atmophsere of 3-7% CO2.
2. Blood culture: Blood samples taken during bacteraemia
(meningococcaemia) may be positive for meningococci.
3. Nasopharyngeal swabs (Wests swabs): The swabs are cultured on
chocolate or Thayer Martin agar and the suspected growth is identified and
differentiated from other commensal neisseria which may be found in the
nasopharynx by the following methods:

V. On what basis N. meningitidis is differentiated from commensal

Neisseria:
N. meningitidis
1. Growth on simple
media (e.g. nutrient agar)
2. Sugar fermentation
3: Agglutination with
Specific antisera

32

Commensal Neisseria

VI. Illustrative pictures for the M.O.

1. Microscopical examination:
a) Neisseria in culture:

2. Culture characteristics:
a) Chocolate agar

3. Biochemical reactions:
a) Oxidase test:

33

7)Family: Enterobacteriaceae
I. Lactose Fermentors
I. Classification:
Escherichia coli
Klebsiella
Citrobacter

II. Morphology and staining reaction:

E. coli:
Gram-negative short rods, motile, non-capsulated, non-spore forming,
aerobic or facultative anaerobes.
Klebsiella:
Gram-negative, capsulated, non-motile and non-spore forming coccobacilli.
Citrobacter:
Similar to E. coli

III. Culture characteristics:

E.coli:
1- Nutrient agar circular, convex and small smooth colonies.
2- MacConkeys medium Rose pink colonies (lactose fermentor).
3- Eosine Methylene Blue agar (E.M.B.) metallic sheen colonies

Klebsiella:
1- Mucoid colonies on nutrient agar or MacConkeys medium.
2- Rose pink colonies on MacConkeys medium (lactose fermenter).

34

IV. Diseases:
E. coli:
1. Wound infection, pneumonia and meningitis (especially in newborns).
2. Urinary tract infection.
3. Endotoxic shock.
4. Faecal pollution of water.
5. Travelers diarrhoea:

Klebsiella:
1- K. pneumoniae that causes pneumonia and urinary tract infection.
2- K. rhinoscleromatis that causes rhinoscleroma, a destructive lesion of
nose and pharynx.
Citrobacter:
Urinary tract infection and sepsis

IV. Illustrative pictures for the M.O.

1. Microscopical examination:
a) Bacilli (in culture)

35

b) Klebsiella (capsule stain)

2. Culture characteristics:
a. MacConkeys media:
Lactose fermentor: Pink colonies

36

b. EMB (Eosin Methylene Blue):

Lactose fermentor: Green metallic sheen

c) TSI (Triple Sugar Iron) agar:

37

3. Biochemical reactions:
a) Sugar fermentation:
Sugar fermentors with production of acid & gas

b) IMViC test:
(i) E.coli

38

(ii) Klebsiella

39

II. Non-lactose Fermenters

Salmonella
Shigella
Proteus
II. Morphology and staining:
Salmonella :
Gram-negative, short, non-capsulated, non-spore forming and facultatively
anaerobic bacilli. Most of them are motile.
Shigella:
Gram-negative, non-motile, non-capsulated and non-spore forming bacilli.
Proteus:
Gram-negative bacilli, characterized by being non-capsulated, non-spore
forming, pleomorphic and highly motile with peritrichous flagella.

III. Culture characteristics:

Salmonella:
They grow on ordinary media and are lactose and sucrose non-fermenters.
Preliminary differentiation between Salmonella typhi and paratyphi
depends on biochemical reactions. Salmonella typhi ferments glucose,
maltose, mannose and mannite with the production of acid only while
Salmonella paratyphi produces acid and gas.
Shigella:
Grow on selenite or tetrathionate broth and MacConkeys or desoxycholate
citrate agar (D.C.A.).
Proteus:
Grows on simple and enriched media producing a concentric wavy growth
(swarming).

40

IV. Diseases and clinical specimens for laboratory diagnosis:

Salmonella:
Members of Salmonella are responsible for two diseases in man:
1. Enteric fever which includes typhoid and paratyphoid fevers. It is a world
wide disease and is endemic in Egypt,
2. Salmonella food poisoning which local intestinal infection is leading to
vomiting and diarrhea.
Shigella:
After the transmission of organism the infection is localized in the
gastrointestinal tract without blood invasion. Shigella spp. produces 2 types
of toxins:
1. Endotoxin i.e. the LPS component of the cell wall, which causes
irritation of the intestinal mucosa.
2. Exotoxin (produced mainly by Shigella dysenteriae): This toxin has
cytotoxic, neurotoxic and enterotoxic action (similar to LT of E. coli).
Neurotoxic effect results in the inhibition of sugar and amino acids
absorption from intestine. The toxin causes ulceration, bleeding and
necrosis of the intestinal mucosa resulting in pseudomembrane
formation on the ulcerated area and presence of blood and pus in
stool.
Proteus:
1- Urinary tract infections.
2- Wound infections.
3- Otitis media.
4- Summer diarrhea.

41

V. Illustrative pictures for the M.O.

1. Microscopical examination:

Gram -ve bacilli

2. Culture characteristics:
a) MacConkeys media:
Non-Lactose fermentor: Pale yellow colonies

42

b) Nutrient agar (swarming growth) (proteus):

c) S-S agar:

43

d) TSI (Triple sugar iron agar):

Salmonella

BACTERIUM

Shigella

SLANT BUTT

H2S COMMENTS

Shigella dysenteriae

Causes food infection dysentery

Salmonella typhimurium

YG

Causes food poisoning

Salmonella typhi

Causes typhoid fever

Aerobacter aerogenes

YG

Similar to Klebsiella,but nonrespiratory

Escherichia coli

YG

Most common of GIflora

Citrobacter freundii

YG

Proteus vulgaris

YG

+ Causes GU infections, highly motile

Klebsiella pneumoniae

R or YG

Pneumonia in debilitated patients

(nosocomial)

Pseudomonas aeruginosa

GI inhabitant, causes wound, GU infections

Alcaligenes faecalis

GI inhabitant, opportunistic pathogen of GU

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one of "paracolon" group (non-pathogenic)

3. Biochemical reactions:
a) IMViC test:

Salmonella

Shigella & Proteus

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b) Urease test:
Proteus ( +ve urease test)

4. Special tests:
a) Widal test for salmonella:

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8)Pseudomonas
I. Morphology and staining reaction:
This group of microorganisms is Gram-negative, motile, non- capsulated and
non-spore forming bacilli.

II. Culture characteristics:

Bluish green coloration on nutrient agar as a result of pigment production.
Haemolysis of blood (some strains).
Sweet grape like odour (some strains).

III. Diseases and clinical specimens:

They are pyogenic bacteria usually causing hospital acquired infections.
Clinical specimens depend on the clinical presentation and the site of
pyogenic lesion:
Disease

Clinical specimen for lab diagnosis

1. Wound sepsis.

2. Urinary tract sepsis

3. Respiratory tract infections:

..

4. Meningitis:

5. Otitis externa:

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IV. Illustrative pictures for the M.O.

1. Microscopical examination (Gram -ve bacilli)

2. Culture characteristics:
a) Pseudomonas on nutrient agar (Diffuse greenish discoloration:
exopigment production)

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b) Triple sugar iron (TSI) agar:

3) Biochemical reactions:
a) Sugar oxidation:
Sugar oxidation with production of acid only.

b) Oxidase test: positive.

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9)Brucella

I. Classification:
1. Brucella abortus (B. abortus):
2. Brucella melitensis (B. melitensis):
3. Brucella suis (B. suis):
4. Brucella canis (B. canis):

A pathogen of cattle
A pathogen of sheep
A pathogen of pigs
A pathogen of dogs

II. Morphology and staining reaction:

These organisms are Gram-negative, aerobic, non-motile, non-capsulated
and non-spore forming short bacilli (cocco bacilli ).

III. Culture characteristics:

The organism can grow on enriched media as blood agar, trypticase soy agar
and media enriched with liver extract.
B. abortus requires 5-10% Co2 for growth while other species can grow in
air.

IV. Lab. diagnosis of Brucellosis:

1. Culture:
Clinical specimens are taken during the first week of the febrile stage (acute
phase of illness), blood sample for blood culture, and bone or lymph node
biopsy for culture on other media.
a. Blood culture: Organisms are present in the blood stream during the
febrile stage. Blood culture is done on liver infusion broth. Duplicate
cultures are carried out: one to be incubated in 5-10% Co2 (for the growth of
B. abortus), and the other is incubated aerobically.
Subcultures are done every few days on liver extract agar. The cultures
should be kept for at least 6 weeks before they are considered negative.

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b. Culture on trypticase soy broth or thionine tryptose broth. Duplicate

cultures are done, one to be incubated in 5-10% Co2 and the other is
incubated in normal atmospheric condition.
Subcultures are done, at several days intervals, on solid media of similar
composition. The cultures should be kept for at least 3 weeks before
considering negative.
V. Lab. diagnosis of Brucellosis:
1. Isolation and identification:

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10) Vibrio
I. Classification:
Vibrio cholerae (V. cholerae) which is classified according to somatic (O)
antigen into 6 main O-groups.

II. Morphology and staining reaction:

V. cholerae is short, motile with a single polar flagellum, Gram- negative,
non-capsulated, non-spore forming, aerobic and curved (comma-shaped)
bacilli.

III. Culture characteristics:

It grows on simple media. Growth can occur in alkaline pH (pH 9). Its
habitat is marine and surface waters (water-borne disease).

IV. Clinical specimens:

I. Diagnosis of a case during an epidemic:
The diagnosis is done by a simple microscopical examination of stools for
the presence of vibrios.
II. Diagnosis of a suspected case in a non-endemic area:
Isolation of the organisms by inoculation of rice watery stool or direct rectal
swab in alkaline peptone water (pH 8.5-9) or Dieudonnes medium (alkaline
blood agar, pH 9-9.6) and incubation at 37C for 6-8 hours.
The high pH prevents the growth of most other intestinal flora and allows
the vibrios to grow forming a surface pellicle. Subcultures are then made on
alkaline agar or thiosulphate citrate bile sucrose agar (T.C.B.S.) on which V.
cholerae produces yellow colonies while other stool flora are suppressed.

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V. Illustrative pictures for the M.O.:

1. Microscopical examination:
Vibrio cholerae (Gram stained)

2. Culture characteristics:
Vibrio cholerae on TCBS
(Yellow colonies)

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Biochemical reactions:
1- Cholerae red reaction:
V. cholerae produces indole and reduces nitrates into nitrities. On
account of these 2 reactions, nitrosoindole compound is produced on nitrate
peptone medium. This shows a red colour with strong acid (sulphuric acid)
2- Indole positive
3. V.P negative
4. Oxidase positive
5. Organism ferments glucose, maltose, mannite and sucrose.

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11) Mycobacterium
I. Classification:
The 2 important modes of infection by tubercle bacilli are milk-borne
infection with the bovine type, and the air-borne infection, generally with the
human type.

II. Morphology and staining reaction:

M. tuberculosis:
The tubercle bacilli are acid and alcohol fast and appear red.
M.leprae:
Films stained with Ziehl-Neelsen show the acid fast bacilli in very large
clumps.

III. Culture:
M. tuberculosis:
Dorsets egg medium or lowenstein-Jensen (aspargine, egg, potato flour,
salts and malachite green) at 37oC in screw-capped universal bottles.

M.leprae :
They are differentiated from Myco. Tuberculosis by their inability to grow
on egg medium or to infect animals

IV. Clinical specimens for laboratory diagnosis:

Pulmonary tuberculosis:
Intestinal tuberculosis:
Urinary tuberculosis:
Tuberculous meningitis:
Leprosy:

sputum
stool
urine
CSF
skin biopsy

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V. Illustrative pictures for the M.O.:

1) Microscopical examination:
a) Tubercle bacilli (Z.N. stain)

b) M. leprae (modified Z.N. stain)

2) Culture characteristics:
M. tuberculosis on Lowenstein Jensen media:

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12)Spirochaetacae
I. Classification:
Contains four human pathogens as well as at least six nonpathogens .

II. Morphology and staining reactions:

A helical shape with tapered ends. The length of the cells was variable,
about 1025 m, and the diameter was 0.20.28 m. Five insertion points of
flagella were observed in the tapered ends Numbers of flagella found were
more than 10 in each end of the cell.
The spirochaete divided by binary fission
III. Culture characteristics:
Special culture media
IV. Illustrative pictures for the M.O.:
1. Microscopical examination:
Spirochaetes

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MYCOLOGY
Fungi of medical importance
Although they are from 50.000-100.000 species of fungi very few of
them are Pathogenic to man and animal
They may attack:
1- The skin and/or mucous membrane (superficial mycoses).
2 - The internal organs as the lungs, central nervous system, bones etc.
(Systemic mycoses).

Superficial Mycoses
The most famous fungi causing superficial fungus infection are
Dermatophytes and Candida. Dermatophytes attack keratinous structures
of the skin horney layer), hair and nail and cause the group of diseases
known as Tinea.
Candida (previously known as monilia) can attack the skin, the mucous
membranes and rarely the internal organs.

Dermatophytes
This is a group of dermatophytes related fungi which attacks the
keratinous structures of the skin and causes diseases known as Tineas.
Diseases or the skin caused by Dermatophytes:
1- Scalp: Tinea capitis (ringworm).
2 - Glabrous skin: Tinea circinata.
3 - Inguino-scrotal and Inguino-labial areas: Tinea cruris.
4 - Feet: Tinea pedis.
5 - Nails: Onychomycosis.
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Classification or Dermatophytes:
They are classified into 3 genera according to the shape of macroconidia
formed in the culture:
a) Microsporum: forms large, Spindle-shaped thick walled macroconidia
which are divided by 5 - 12 transverse septa.
b) Trichophyton: forms thin walled, smooth macroconidia which are
either spindleshaped or cylindrical. They are divided by 4 - 6
transverse septa.
c) Epidermophyton; forms pear-shaped thick walled, smooth
macroconidia with 3 transverse septa.
Some relevant information about dermatophytes:
1 - Microsporum affects skin and hair, Trichophyton affects skin, hair
and nail, while Epidermophyton affects skin and nail but not hair.
2 - Each geographic locality has its own dermatophyton e.g.
Trichophyton violacium is the prevalent causative organism of Tinea
capitis in Egypt while Microsporum auduini is the prevalent cause in
England.
3 - Some Dermatophytes affect man only and are known as
Anthropophilic others affect animals mainly and can be transmitted to
man (zoophilic organisms) and a third group live in soil and can affect
man (Geophilicorganisms).
4 - All Dermatophytes are sensitive to Griseofulvin.

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Candida
Candida is a yeast-like organism that lives as a commensal in oral
mucosa, intestine and vagina of women.
It is an opportunistic organism and can attack skin and/or mucous
membranes under certain conditions.
Rarely it attacks internal organs in immunosuppressed persons.
Candida albicans is the most common pathogen of the Candida group.
Factors which predispose to infection with Candida aIbicans:
1 - Prolonged courses of broad spectrum antibiotic.
2 - Diabetes Mellitus
3 - Prolonged courses of corticosteroid therapy.
4 - Immunosuppressive drugs.
5 - Contraceptive pill.
6 - Local factors in the skin as increased hydration of the horny layer.
Diseases caused by Candida albicans (Candidiasis):
I - In the skin :
1 - Candidiasis of the groin.
2 - Interdigital candidiasis.
3 - Candidiasis of toe webs
4 - Chronic Paronychea. .
5 - Angular Stomatitis.

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II- In the mucous membranes:

1 - Oral thrush.
2 - Glossitis.
3 - Candidal vaginitis.
4 - Candidal enteritis.
III- Internal organs (rare):
1 - Bronchopneumonia.
2 - Endocarditis.
Treatment of candidiasis:
Candida responds to Nystatin and Amphoterecin B but not to
Griseofulvin.

Recently broad spectrum antimycotics have been

discovered which are effective against Dermatophytes, Candida and

some organisms causing systemic mycoses e.g. Imidazole group. There
are local imidazole preparations of imidazole as clotrimazol (canesten)
Miconazole (dactarine) or systemic imidazole as Ketoconazole (nizoral
tables).

Diagnosis of Superficial Mycoses

1 - Direct microscopic examination:
The lesions are scraped with a blunt scalpel; the collected scales are put
onto a slide, add a drop of 15% KOH, put a cover slide, warm gently and
leave for 20 minutes. In positive cases hyphae, spores or budding cells
will be seen.
2 - Culture on Sabouraud's Dextrose agar to which antibiotic is added to
suppress bacterial growth.The culture is incubated at room temperature

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(22C) observed daily for 2 - 3 weeks. Identification is done by the

characters of the colony, its colour, its rate of growth and also by
microscopic examination of a part of the colony.
3 - In skin biopsy fungi do not appear in sections stained by
haematoxylin and eosin, but can be demonstrated in sections stained with
PAS. ( The periodic acid-Schiff).

Systemic Mycoses
It comprises a group of diseases which affect mainly the internal organs
and can affect secondarily the skin.
They are rare in Egypt and therefore a few examples of them will be
mentioned:
- Blastomycosis caused by Blastomyces dermatitidis affecting primarily
the lungs but may disseminate to affect the skin, bones, CNS and sites.
- Cryptococcosis caused by Cryptococcus neoformans affecting mainly
the brain and meningese and also the lungs.
- Histoplasmosis caused by Histoplasma capsulatum affecting primarily
the lungs and involves also the spleen, liver and kidney.

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Diagnosis of deep fugal infection:

1 - Direct examination of the exudate.
2 - Culture both on sabouraud agar incubated at 22C and on blood agar
incubated at 37C this is done to detect Dimorphic organisms which will
grow as filamentous colonies in the first case and as yeast forms in the
second case

e.g. Blastomyces dermatitidis.

3 - Demonstration of organisms in tissue sections stained with PAS.

4 - Intradermal tests with antigens extracted from cultures of organisms
e.g. Blastomycin or Histoplasmin.
5 - Serological tests as CFT using extract of fungus as the antigen.

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Tinea capitis (ringworm)

Tinea circinata

Tinea cruris

Tinea pedis

Onychomycosis

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Interdigital candidiasis

Angular Stomatitis

Oral thrush

Glossitis

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Blastomyces dermatitidis

Cryptococcus neoformans

Histoplasma capsulatum

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I. BACTERIAL SEROLOGICAL TESTS

1- AGGLUTINATION REACTIONS
The antigen:
in agglutination reactions is particulate rmd commonly consists of
suspensions of microorganisms, cells (red blood cells) or uniform particles
like latex on which antigens have been adsorbed.
The reaction:
when the antigen is mixed with antiserum, clumps aggregate and settle as
large visible clumps.
Procedure:
1. Slide agglutination:
A drop of a particulate antigen is mixed with a drop of antibody. Mix well
and observe for agglutination within few minutes.

Slide agglutination test

2. Tube agglutination:
The reactants are mixed in a tube and after incubation at suitable
temperature for a certain time, clumps of antigen particle will be seen at the
bottom of the tube. For estimation of the amount of antibody (antibody titer)
a serial dilutions of the antibody are mixed with a fixed amount of antigen.
After incubation, the antibody titer is determined as: The highest dilution of
the antibody which shows agglutination.

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Applications of agglutination reaction:

1. Blood grouping:

Blood grouping
2. Identification of unknown organism:
If an organism is isolated from a patient, a drop of saline suspension of this
organism is mixed with known antibody on a slide, if agglutination occurs,
then this organism will be identified according to the known antibody.
3. Identification of unknown antibody:
This is the reverse of the previous example, where unknown antibody is
mixed with known organism. It could be done quantitatively to determine
the antibody titer.
A common example is: Widal test for diagnosis of typhoid fever.
Steps:
1- Serial dilutions of patient serum are mixed with fixed amount of
salmonella antigen.

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2 - Determine the antibody titer as the highest dilution of antibody showing

agglutination.

Widal test for diagnosis of typhoid fever

4. Test for pregnancy:
Principle:
During pregnancy the hormone chorionic gonadotrophin is excreted in urine.
As the hormone is soluble, it is adsorbed on a particle (latex particle) to render
agglutination visible.

Pregnancy test
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5. Diagnosis of Rh incompatibility:
This test aims to detect Rh antibody in mother's serum.
Direct coomb's test:
Mix serum (antibody unknown) of the mother with Rh + ve cells (groupO).
If agglutination occurs

There is Rh antibody in mother's serum

Indirect coomb's test:

Sometimes Rh antibodies are incomplete, (monovalent) so they cannot
agglutinate particles but can bind to them firmly. It could be carried out in 2
steps;
a. Serum of the patient + Rh - Ve cells (group O )
b. Add antihuman globulin

visible agglutination.

N.B. We consider the Rh antibody as an antigen for the second step as it is

globulin.

Coomb`s test
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2-PRECIPITATION REACTION
The antigen in this reaction is in antibody, when it is mixed with the antibody
precipitation will occur when they are at their optimum concentrations.
If either the antibody or the antigen is present in excess, the precipitation is
not complete. This phenomenon is called the zone phenomenon.
Methods:
1. Capillary tube ring test:
A solution of the antiserum containing the antibody is withdrawn in a
capillary tube. Then the soluble antigen is added and makes contact with the
antiserum. After a period of time, if the antiserum contains specific antibody
against the antigen, a ring of precipitate forms at the point of the optimum
concentration of both reactants. e.g. in grouping of streptococci.

Capillary tube ring test

2. Single diffusion test:
Antiserum is incorporated into a layer of agar in the bottom of a tube, and the
antigen is layered over the agar, as the antigen diffuses into the agar, a
precipitate forms at a position where the reactants are at the optimum
concentration.
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3. Radial immumodiffusion:
A plate of agar is poured and wells (holes) are cut into the agar. The antiserum
is incorporated in the agar and antigens in the wells. As the antigen and
antibody diffuse toward each other, a precipitate forms which is visible as a
definite ring in the agar.

Radial immunodiffusion
Applications of precipitation reactions:
1. Diagnosis of rheumatic fever or other degenerative diseases i.e. Creactive protein
Procedure:
-Serum of the patient is mixed in a capillary tube with anti C-reactive
protein antibody. Appearance of precipitate means positive reaction.
- The amount of precipitate is proportionate to the amount of C-reactive
protein in the serum.
2. Antigenic classification of bacteria.
3. In medico legal tests, i.e. identification of blood stains.

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3- TOXIN-ANTITOXIN NEUTRALIZATION
a. Antistreptolysin O test:
In the test, commercially available streptolysin O in a standard buffer is
mixed with dilutions of the patient's serum and incubated.
A saline suspension of human or rabbit red blood cells is then added to each
dilution tube. If the patient has been or is in contact with group A
streptococci, specific antibodies ( antistreptolysin O) will be present to
neutralize the streptolysin O in the first step of the test.

Thus the red blood

cells that are added later will not lyse in these tubes. If no antibodies are
present, haemolysis will occur.
Antistreptolysin O titer:
is the last tube showing no haemolysis and is expressed as Todd units.
a titer of 200 Todd units is normal.

Anti-streptolysin-O test

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b. Elek's test:
It is used to determine whether or not an isolated strain of C. diphtheriae is
toxigenic.
Method:
A strip of filter paper with antitoxin impregnated in it is placed into melted
agar and allowed to be trapped in the agar as it cools.
The isolated strain of the suspected bacterium is streaked on the agar surface
at 90 angles to the strip.
If the organism produces toxin, lines of precipitation will be formed at the
region where the antitoxin and soluble toxin meet to form a precipitate.

Elek`s test

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4. COMPLEMENT FIXATION TEST (CFT)

A complement is a protein serum which can fix itself nonspecifically to an
antigen-antibody complex. When an antigen is a cell, lysis of the cell will
occur as a result of this fixation of the complement. This process is called
complement-fixation. The complement-fixation test is a widely used
serological test for the diagnosis of many infectious diseases that result from
the activities of bacteria, rickettsiae and viruses.
One example of a complement fixation test is Wasserman test for syphilis. In
this test an extract of a beef heart that contains a complex of phospholipids
called cardiolipin is used as the antigen instead of the spirochetes
Treponema pallidum.
Precautions to be taken during the test:
1. Adjust the amount of the complement.
2. RBC s are suspended in saline to avoid haemolysis by distilled water.
3. Adjust the amount of the antigen.
4. Antigen control and serum control are included in the test to detect the
anticomplementary activity of either antigen or serum.

The haemolysis of RBCs

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5. FLUORESCENT ANTIBODY TECHNIQUES

If an antibody is labeled with a fluorescent dye (fluorescein
isothiocyanate) and exposed to ultraviolet light, this dye produces
visible fluorescent light and is seen as a bright spot. Two types of
fluorescent antibody tests:
1. Direct fluorescent antibody test
2. Indirect fluorescent antibody test

Direct fluorescent antibody test

Indirect fluorescent antibody test

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Fluorescent microorganisms stained by the fluorescent antibody techniques

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6- RADIOIMMUNOASSAY(RIA)
Antigen in saline is incubated on a plastic plate or tube. Small quantities
become absorbed onto the plastic surface and free antigen is washed away.
Test antibody is added which binds to the antigen. Unbound proteins are
washed away.
The antibody is detected by a radiolabeled legend. Unbound legend is
washed away and the radioactivity of the plate is counted on a gamma
counter.
A titration curve is plotted. With increasing amounts of test antibody, the
counts per minute (cpm) rise through a linear range to a plateau
Applications:
Quantitation of hormones (insulin), metabolics (folic acid), drugs
(morphine), microbial antigen (e.g. hepatitis B antigen) and serum proteins
(e.g. carcinoembryonic antigen).

Radioimmunoassay

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7-ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

It is an antigen-antibody reaction with the same principle as RIA.
The difference is that in ELISA, the antibody is labeled with an enzyme
instead of the radioactive isotope.

Enzyme Linked Immunosorbent Assay (ELISA)

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