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Medical Microbiology
PM 301-p
For
Third Year Pharmacy Students
By:
Prof. Dr. M.Seif Eldin Ashour
2006-2007
Acknowledgement
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
2ndweek
3rd week
4th week
5th week
6th week
7th week
8th week
9th week
10th week
Mycology
11th week
Agglutination reactions
12th week
13th week
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
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.
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.
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.
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.
1) Staphylocoocus
I) Classification according to endopigment production:
1. Staphylococcus aureus
2. Staphylococcus epidermidis
(Staphylococcus albus)
white pigment.
3. Staphylococcus saprophyticus
2. Culture characteristics:
a) Nutrient agar
White colonies
....
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
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
*
*
Post-streptococcal diseases:
*
*
*
Rheumatic fever.
Glomerulonephritis.
Erythema nodosum.
13
2. Culture characterstics:
a) Blood agar plate showing:
- haemolysis: ................
-haemolysis: ..........
-(no haemolysis): ...............
3. Biochemical reactions:
a) Bacitracin sensitive strain:
(Strept. .)
14
Strept.
Viridan
Streptococcus
Pneumoniae
c) Bile solubility test:
Principle:
Reading of the test:
15
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
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
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
Unprotected animal
Toxigenic strain
Non-toxigenic strain
Antitoxin protected
animal
Survival
Survival
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.
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
23
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
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.
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.
30
32
Commensal Neisseria
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
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
35
2. Culture characteristics:
a. MacConkeys media:
Lactose fermentor: Pink colonies
36
37
3. Biochemical reactions:
a) Sugar fermentation:
Sugar fermentors with production of acid & gas
b) IMViC test:
(i) E.coli
38
(ii) Klebsiella
39
40
41
42
c) S-S agar:
43
Salmonella
BACTERIUM
Shigella
SLANT BUTT
H2S COMMENTS
Shigella dysenteriae
Salmonella typhimurium
YG
Salmonella typhi
Aerobacter aerogenes
YG
Escherichia coli
YG
Citrobacter freundii
YG
Proteus vulgaris
YG
Klebsiella pneumoniae
R or YG
Pseudomonas aeruginosa
Alcaligenes faecalis
44
3. Biochemical reactions:
a) IMViC test:
Salmonella
45
b) Urease test:
Proteus ( +ve urease test)
4. Special tests:
a) Widal test for salmonella:
46
8)Pseudomonas
I. Morphology and staining reaction:
This group of microorganisms is Gram-negative, motile, non- capsulated and
non-spore forming bacilli.
1. Wound sepsis.
..
4. Meningitis:
5. Otitis externa:
47
2. Culture characteristics:
a) Pseudomonas on nutrient agar (Diffuse greenish discoloration:
exopigment production)
48
3) Biochemical reactions:
a) Sugar oxidation:
Sugar oxidation with production of acid only.
49
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
50
51
10) Vibrio
I. Classification:
Vibrio cholerae (V. cholerae) which is classified according to somatic (O)
antigen into 6 main O-groups.
52
2. Culture characteristics:
Vibrio cholerae on TCBS
(Yellow colonies)
53
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.
54
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.
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
sputum
stool
urine
CSF
skin biopsy
55
2) Culture characteristics:
M. tuberculosis on Lowenstein Jensen media:
56
12)Spirochaetacae
I. Classification:
Contains four human pathogens as well as at least six nonpathogens .
57
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.
58
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.
59
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.
60
61
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.
62
63
Tinea circinata
Tinea cruris
Tinea pedis
Onychomycosis
64
Interdigital candidiasis
Angular Stomatitis
Oral thrush
Glossitis
65
Blastomyces dermatitidis
Cryptococcus neoformans
Histoplasma capsulatum
66
67
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.
68
Pregnancy test
69
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
visible agglutination.
Coomb`s test
70
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.
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.
72
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.
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
73
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
74
76
78
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
79
80