Handbook of Practical Examination in Microbiology Neeta Patwardhan, Sharad Digambar Bhat, Satish Patwardhan
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Microscopic SlidesCHAPTER 1

 
STAPHYLOCOCCI
Q1. Identify and describe (Fig. 1).
Ans. Slide of Staphylococcus—These are gram positive cocci arranged in grape like clusters, non-motile, non-sporing approximately 1 µ in diameter.
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Figure 1: Staphylococci
Q.2. Mention its pathogenicity.
Ans.
  1. Cutaneous lesions:
    • Carbuncles
    • Styes
    • Boils
    • Abscess
    • Impetigo
    • Pemphigus neonatorum
    • Sepsis
    • Wound infection and infection in burns
    • Conjunctivitis.
  2. 2Deep infections:
    • Bone and joint
      • Acute osteomyelitis
      • Pyoarthritis/Septic arthritis
    • Cardiac—Acute bacterial endocarditis, pericarditis
    • CNS—Meningitis, brain abscess
    • Kidney—Renal abscess
    • Intestinal—Enterocolitis
    • Bloodstream—Septicemia, pyloric abscess.
  3. Toxin associated infections:
    • Toxic shock syndrome
    • Staphylococcal scalded skin syndrome (SSSS)
    • Food poisoning.
  4. Iatrogenic infections.
Q.3. How it is classified?
Ans.
  1. Classification based on pigment production:
    • Golden yellow pigment—S. aureus
    • White pigment—S. albus
    • Lemon yellow pigment—S. citreus.
  2. Classification based on coagulase production:
    • Coagulase positive
      • Staphylococcus aureus (pathogenic to human)
      • Staphylococcus intermedius (animal strain, not pathogenic to human)
      • Staphylococcus hyicus (animal strains, not pathogenic to human).
    • Coagulase negative
      • Staphylococcus epidermidis
      • Staphylococcus saprophyticus
      • Staphylococcus haemolyticus.
Q.4. Mention selective media for its isolation.
Ans.
  • Mannitol salt agar
  • Ludlam's medium
  • RCM with 10% sodium chloride.
Q.5. Mention its pathogenicity markers.
Ans.
  • Coagulase positive
  • Fermentation of mannitol anaerobically
  • Beta hemolysis
  • Golden pigmentation
  • Phosphatase positive
  • Hydrolyze urea
  • Gelatin liquefaction
  • Black color colonies on BPT.
Q.6. Mention various toxins produced by it.
Ans.
  1. Cytolytic toxins:
    • Hemolysins
    • Leukocidins (Panton Valentine toxin).
  2. Enterotoxin.
  3. 3Exfoliative toxin—Epidermolytic toxin.
  4. Toxic shock syndrome toxin (TSST).
Q.7. Mention various enzymes produced by it.
Ans.
  • Coagulase
  • Phosphatase
  • Deoxyribonuclease
  • Hyaluronidase
  • Staphylokinase
  • Lipases
  • Proteases.
Q.8. Which are the antibiotics commonly used to treat infections with this organism?
Ans.
  • Benzyl penicillin—most effective in sensitive strains
  • Cloxacillin—used against beta lactamase producing strains
  • Vancomycin and Teicoplanin—for MRSA strains
  • 1st generation and 2nd generation cephalosporins can be used
  • Clindamycin—useful in osteomyelitis
  • Bacitracin, chlorhexidine and mupirocin—for mild superficial lesions (topical application).
Q.9. Which is the common site of carriage of this organism in healthy people and percentage of carriage?
Ans. About 30–50% people carry the organism. The most common site in anterior nares followed by groin, perineum and throat.
 
STREPTOCOCCI
Q.1. Identify and describe (Fig. 2).
Ans. Slide of streptococci—They are gram positive cocci, spherical or oval, 0.5–1.0 m in size and arranged in chains.
zoom view
Figure 2: Streptococcus
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Q.2. How they are classified?
Ans.
  1. Classification based on O2 requirement:
    • Aerobes—facultative anaerobes
    • Obligate anaerobes—peptostreptococci.
  2. Classification based on type of hemolysis of RBC's produced on blood agar:
    • Alpha hemolysis—Streptococcus viridans
    • Beta hemolysis—Beta hemolytic streptococci
    • Gamma hemolysis (No hemolysis)—Enterococcus group
  3. Classification based on the nature of carbohydrate (C) antigen on the cell wall (Lancefield groups):
    • Beta hemolytic streptococci are classified into 20 groups named A to V (except I and J)
    • Streptococcus pyogenes belongs to Group A.
Q.3. Mention pathogenicity of Streptococcus pyogenes.
Ans.
  1. Respiratory infections:
    • Sore throat
    • Pharyngitis
    • Tonsillitis
    • Scarlet fever.
  2. Skin infections:
    • Impetigo
    • Erysipelas.
  3. Genital infections:
    • Puerperal sepsis
  4. Other suppurative infections:
    • Infection of skin lesions—wounds, burns, scabies, eczema, psoriasis
    • Abscesses in internal organs—e.g. brain, liver, kidney
    • Septicemia
    • Pyemia
    • Streptococcal toxic shock syndrome.
  5. Non-suppurative lesions produced by it
    • Acute rheumatic fever
    • Acute glomerulonephritis.
Q.4. Mention names of the serological tests used for its retrospective diagnosis.
Ans.
  • ASO test—for rheumatic fever
  • Anti-DNAse B (Anti-Deoxyribonuclease B) test—for glomerulonephritis
  • Anti hyaluronidase test
  • Streptozyme test.
Q.5. Mention some important toxins and enzymes produced by this organism.
Ans. Toxins:
  1. Hemolysins:
    • Streptolysin ‘O’
    • Streptolysin ‘S’
  2. Pyrogenic toxin (Erythrogenic toxin)—now renamed as, “Streptococcal pyrogenic exotoxin” (SPE).
Enzymes:
  1. Streptokinase (Fibrinolysin).
  2. 5Deoxyribonuclease (Streptodornase).
  3. Nicotinamide adenine dinucleotidase (NADase).
  4. Hyaluronidase.
Q.6. Which is the selective medium for this organism?
Ans. BA containing 1:1,000,000 crystal violet and 1:16,000 sodium azide (Pike's medium)—permits growth of streptococci but inhibits growth of other bacteria specially staphylococci.
Q.7 Which tests are used for the confirmation of Streptococcus pyogenes?
Ans.
  1. Bacitracin sensitivity testing. Streptococcus is sensitive to bacitracin.
  2. PYR test-It is PYR test positive
 
PNEUMOCOCCI (STREPTOCOCCUS PNEUMONIAE)
Q.1. Identify and describe (Fig. 3).
Ans. Slide of Pneumococcus: These are gram positive, small (1 m in diameter), slightly elongated cocci arranged in pair (diplococci) with the broad ends in opposition, presenting a flame shaped /lanceolate appearance. They are capsulated.
zoom view
Figure 3: Pneumococcus
Q.2. Mention its pathogenicity.
Ans.
  • Lobar pneumonia
  • Bronchopneumonia
  • Commonly associated with the acute exacerbation in chronic bronchitis
  • Meningitis
  • Suppurative lesions—Empyema, pericarditis, otitis media, sinusitis, conjunctivitis, suppurative arthritis and peritonitis.
Q.3. Mention the media used for its cultivation.
Ans. Blood agar, 5–10% CO2 improve its growth.
6
Q4. How is it differentiated from Streptococcus viridans?
Ans. It is differentiated as shown in following table
Characters
Pneumococci
Steptococcus viridans
Morphology
Capsulated, lanceolate, diplococci
Non-capsulated oval or round cells in chains
Quellung test
Positive
Negative
Colony morphology
Initially dome shaped with a hemolysis, later on ‘draughtsman’ colonies
Dome shaped with a-hemolysis
Growth in liquid media
Uniform turbidity
Granular turbidity
Bile solubility
Positive
Negative
Inulin fermentation
Positive
Negative
Optochin sensitivity
Positive
Negative
IP inoculation in mice
Fatal infection
Non-pathogenic
Q5. What are the characteristic features of the colony of this organism?
Ans. Initially, the colony is dome shaped. On further incubation, it becomes flat with raised edges and central umbonation. When viewed from above, concentric rings are seen, i.e. draughtsman appearance or carrom coin appearance.
In aerobic condition, they show alpha hemolysis and in anaerobic condition, they show beta hemolysis.
Q6. What is Quellung reaction? What was its importance in the past?
Ans. It is also known as the ‘capsule swelling reaction’. When a suspension of pneumococci is mixed on a slide with a drop of type specific antiserum and a loopful of methylene blue solution, the capsule appears apparently swollen, sharply delineated and refractile. This test can be done directly with sputum in acute pneumonia cases.
It was used as a bedside procedure in the past when antiserum was used for treatment of pneumonia.
Q7. What is CRP?
Ans. It is an abnormal protein (beta globulin) that precipitates with the somatic ‘C’ antigen of pneumococci and appears in the acute phase sera of cases of pneumonia but disappears during convalescence. Actually, it is not an antibody produced as a result of pneumococcal infection, but is an acute phase substance produced by the hepatocytes. It is produced during bacterial infections, inflammation, malignancies and tissue destruction. It disappears when the inflammatory reactions subside.
Q8. How do you test for presence of CRP?
Ans. It can be detected by a passive agglutination test using latex particles coated with anti-CRP antibody.
Q9. How will you proceed for laboratory diagnosis of pneumonia/other pneumococcal infections?
Ans.
  1. Sample: Sputum, serum-coated laryngeal swabs in infants, respiratory secretions (for pneumonia), CSF for meningitis, fluid aspirated from middle ear in otitis media.
  2. Laboratory diagnosis:
    For Pneumonia
    7Smear of sputum-stained with Gram stain—Gram positive diplococci seen.
    • Culture: Sputum inoculated on blood agar after homogenization and incubated at 37°C under 5–10% CO2. Colony characters noted-draughtsman colony and confirmed by biochemical tests, e.g. Bile solubility, optochin sensitivity and inulin fermentation.
    • Mouse inoculation: If organisms are scanty intraperitoneal inoculation in mouse can be done and pneumococci demonstrated in the peritoneal exudates and heart blood of the mouse after its death (usually within 3 days)
    • Blood culture: In acute stage of pneumonia, organisms may be isolated from blood. Isolation of S. pneumoniae from blood indicates bad prognosis.
    For meningitis
    • Organisms can be demonstrated directly in Gram stained films of CSF (after centrifugation), gram positive diplococci are seen both inside the polymorphs and extracellularly.
    • Diagnosis is confirmed by culture and biochemical tests, e.g. Bile solubility, optochin sensitivity and inulin fermentation.
    Other tests for diagnosis:
    1. Detection of antigen by (especially in meningitis)
      1. Demonstration of SSS in CSF by precipitation with antisera.
      2. Capsular polysaccharide—demonstrated in blood, urine and CSF by counter immuno-electrophoresis.
      3. Other tests like agglutination, precipitation—for detection of antibodies.
      4. Indirect hemagglutination, indirect FA test and RIA have also been employed.
    2. Biomarkers—CRP and procalcitonin.
    3. Molecular methods—PCR have potential when patient has taken antibiotics.
 
GONOCOCCI (NEISSERIA GONORRHOEAE)
Q.1. Identify and describe (Fig. 4).
Ans. Slide of Gonococci: These are gram negative oval cocci arranged in pairs (diplococci) with adjacent sides concave (bean shaped or kidney shaped). Size is 0.5–1 m. They are intracellular within polymorphs (in smears from pus) some cells containing as many as hundred cocci.
zoom view
Figure 4: Gonococci
Q.2. Mention its pathogenicity.
Ans.
  • Urethritis
  • 8Complications:
    • In male—epididymitis, prostatitis, sterility
    • In female—pelvic inflammatory disease and subsequent sterility
  • Rectal infections—proctitis
  • Gonococcal pharyngitis
  • Gonococcal vulvovaginitis in prepubertal girls
  • Disseminated infections—arthritis, rarely meningitis, ulcerative endocarditis
  • Endocarditis
  • Ophthalmia neonatorum—newborn.
Q.3. Name some important samples collected from a patient infected with this organism.
Ans.
  • Urethral discharge
  • Cervical discharge/swab
  • Exudate obtained after prostatic massage
  • Urine (centrifuged).
Q.4. Name the transport medium used to collect samples from patients infected with this organism.
Ans. Charcoal impregnated swabs transported to the laboratory in Stuarts transport medium.
Q.5. Mention two culture media used for its isolation.
Ans.
  • Chocolate agar
  • Thayer Martin medium (containing vancomycin, colistin, nystatin)
  • Chacko Nair egg enriched medium
  • New York city medium
  • Mueller-Hinton agar.
 
CORYNEBACTERIUM DIPHTHERIAE (GRAM's STAIN)
Q.1. Identify and describe (Figs 5 and 6).
Ans. These are thin slender gram positive bacilli approximately 3–6 m by 0.6–0.8 m. They have metachromatic granules at one or both ends and hence appear club-shaped. The bacilli are usually seen in angular fashion resembling the letters V or L. This is called as ‘Chinese letter’ or ‘Cuneiform arrangement’. This arrangement is because of incomplete separation of daughter cells after cell division.
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Figure 5: C. diphtheriae—Gram's stain
zoom view
Figure 6: C. diphtheriae—Albert's stain
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Q.2. Mention few other stains used for staining.
Ans.
  • Loeffler's methylene blue
  • Albert's stain
  • Neisser's stain
  • Ponder's stain.
Q.3. How do the bacilli appear with Albert's stain?
Ans. The bacilli appear green and the metachromatic granules appear bluish black.
Q.4. Mention two media used for culture.
Ans.
  • Blood agar
  • Loeffler's serum slope
  • BPT (Blood Potassium Tellurite agar, Tellurite Blood agar).
Q.5. Which vaccine is used for its prevention? Mention immunization schedule.
Ans.
  1. DPT is used for its prevention.
  2. Immunization schedule:
    • First dose (DPT1)
    • Second dose (DPT2)
    • Third dose (DPT3)
    • Fourth dose (DPT)
    • Fifth dose (DT)
    • 6 weeks
    • 10 weeks
    • 14 weeks
    • 16 to 24 months
    • At the age of 5 years or 6 years.
 
CLOSTRIDIUM PERFRINGENS (CL. WELCHII)
Q.1. Identify and describe (Fig. 7).
Ans. Slide of Clostridium perfringens (Cl. welchii)
These are large stout gram positive bacilli with straight parallel sides and rounded or truncated ends measuring 4–6 m by 1 m with subterminal spores. But spores are rarely seen in culture. They are capsulated and non-motile.
zoom view
Figure 7: Clostridium perfringens
Q.2. Mention the toxins produced by it.
Ans.
  • Alpha
  • Gamma
  • Kappa
  • Beta
  • Eta
  • Lamda
  • Epsilon
  • Delta
  • Mu
  • Iota
  • Theta
  • Nu
(Alpha, Beta, Epsilon and Iota are the four major toxins responsible for pathogenicity)
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Q.3. Mention pathogenicity.
Ans.
  • Gas gangrene
  • Food poisoning
  • Gangrenous appendicitis—intestinal obstruction
  • Necrotizing enteritis
  • Necrotizing colitis
  • Biliary tract infection
  • Brain abscess and meningitis
  • Panophthalmitis
  • Thoracic infection
  • Urogenital infection.
Q.4. Mention the method of cultivation.
Ans.
  • Specimen collection and transport in RCM medium
  • Inoculation on blood agar
  • Incubation in anaerobic condition by using:
    • McIntosh and Filde's jar
    • Gaspak jar
    • Closed glove cabinet.
 
CLOSTRIDIUM TETANI
Q.1. Identify and describe (Figs 8 and 9).
Ans. Slide of Clostridium tetani: Showing gram positive slender bacilli (4–8 µ by 0.5 µ in width) with spherical terminal bulging spores giving the bacillus a drumstick appearance.
zoom view
Figure 8: Clostridium tetani
zoom view
Figure 9: Clostridium tetani
Q.2. Mention the laboratory animal used for laboratory diagnosis.
Ans. Mouse.
Q.3. Mention the vaccines used for active and passive immunization.
Ans.
  1. Active immunization:
    • DPT
    • TT
  2. 11Passive immunization:
    • ATS (Horse, Bovine, Ovine antitetanus serum)
    • ATG (Human antitetanus immunoglobulin).
  3. Immunization schedule—In pregnancy (Tetanus toxoid):
    • First dose—16–36 weeks
    • Second dose—1 month after the first dose.
Note: It is ideal to complete second dose, 3 months before delivery, if not possible then at least one month before delivery by keeping interval of one month between first and second dose.
In Newborn:
  • DPT1
  • DPT2
  • DPT3
  • DPT
  • DT
  • TT
  • First dose
  • Second dose
  • Third dose
  • Fourth dose
  • Fifth
  • 10 years and 16 years
  • 6 weeks
  • 10 weeks
  • 14 weeks
  • 16 to 24 months
  • at the age of 5 years to 6 years
Q.4. Mention toxins produced by it.
Ans. Tetanolysin, tetanospasmin.
Q.5. How is it cultured?
Ans.
  • Sample is collected in RCM
  • Inoculate on BA and incubate by any anaerobic method.
 
MYCOBACTERIUM TUBERCULOSIS (AFB)
Q.1. Identify and describe (Fig. 10).
Ans. Slide of Mycobacterium tuberculosis: These are slender, straight or slightly curved bacilli with rounded ends, occurring singly, in pairs or in small clumps. Size is 1–4 m by 0.2–0.8 m. They are acid-fast, non-capsulated, non-sporing and non-motile, beaded or barred forms are often seen.
zoom view
Figure 10: Mycobacterium tuberculosis
12
Q2. How it is classified?
Ans. Mycobacteria are classified into three groups
  1. Obligate parasites
  2. Opportunistic pathogens
  3. Saprophtyes
    • Obligate parasites are:
      1. Mycobacterium tuberculosis complex which include:
        1. Mycobacterium tuberculosis—human type
        2. Mycobacterium bovis—bovine type
        3. M. africanum—causing human tuberculosis in Africa
        4. M. microti—Pathogenic for voles and other small mammals.
          (Recently three more species have been added: M. canetti (similar to M. africanum), M. caprae (another cattle pathogen) and M. pinnipedii (pathogen of seals)
      2. Lepra bacilli
        Human—M. leprae.
    • Opportunistic pathogens
      Non-tuberculous mycobacteria (NTM)
      1. Photochromogens
      2. Scotochromogens
      3. Nonphotochromogens
      4. Rapid growers.
    • Saprophytic mycobacteria
      1. M. butyricum
      2. M. phlei (Grass)
      3. M. sterocoris
      4. M. smegmatis (Smegma) and other rapidly growing mycobacteria which frequently contaminate urine cultures.
Q.3. Mention its neurological complications.
Ans. Tuberculous meningitis.
Q.4. Mention the media used for its culture.
Ans.
  1. Solid Media
    • LJ medium—egg based
    • Dorset egg agar—egg based
    • Agar based:
      • Middle brook 7H10
      • Middle brook 7H11
    • Loeffler's serum slope—serum based
    • Pawlowsky's medium—potato based
    • Petragnani medium—egg based
    • Tarshis medium—blood based.
  2. Liquid Media:
    • Middlebrook 7H9 medium
    • Sula's and Sauton's medium
    • Proskauer and Beck's medium
    • Dubo's medium
    • Bactec 12 B medium
    • Septi-Chek AFB
    • Mycobacteria Growth Indicator Tube (MGIT).
13
Q.5. Mention different biochemical tests used for its differentiation.
Ans.
  • Niacin test
  • Aryl sulphatase test
  • Catalase-Peroxidase test
  • Neutral red test
  • Amidase test
  • Nitrate reduction test
  • Tween–80 hydrolysis test
  • Tellurite reduction test
  • Susceptibility to pyrazinamide.
Q.6. Which animal is most susceptible to it?
Ans.
  • Guinea pig
  • Hamster.
Q.7. Mention the period required for its growth on conventional media (LJ medium).
Ans. Usually grows within 2–3 weeks, but if there is no growth then wait up to 6–8 weeks.
 
MYCOBACTERIUM LEPRAE
Q.1. Identify and describe (Fig. 11).
Ans. Slide of Mycobacterium leprae—M. leprae is slender, slightly curved or straight bacilli 1–8 m by 0.2–0.5 m. They are acid-fast but less so than M. tuberculosis. They are seen singly or as groups intracellularly or lying free outside the cells. Inside the cell they are seen in the form of bundles called as globi. These globi are present in Virchow's lepra cells or foamy cells which are large undifferentiated histiocytes. In tissue sections, the bacilli are arranged in clumps resembling cigarette ends.
zoom view
Figure 11: M. leprae
Q.2. Mention the classification of the disease.
Ans. It causes leprosy.
  1. It is classified into four types (Madrid classification 1953):
    • Lepromatous
    • Tuberculoid
    • Dimorphous
    • Indeterminate.
  2. 14Ridely and Jopling (1966) classified leprosy into five groups:
    • Tuberculoid (TT)
    • Borderline tuberculoid (BT)
    • Borderline (BB)
    • Borderline lepromatous (BL)
    • Lepromatous (LL).
Q.3. How will you cultivate this organism?
Ans. It is not possible to cultivate lepra bacilli in bacteriological media or in tissue culture, but they can be cultivated by inoculation in foot pad of mice or an nine-banded armadillo.
Q.4. Mention the intradermal test and its uses.
Ans. Use of lepromin test
  • To classify the lesions of leprosy patients
  • To assess the prognosis and response to treatment
  • To assess the resistance of individuals to leprosy
  • To verify the identity of candidate leprae bacilli.
Q.5. Mention laboratory animals used for its study.
Ans.
  • Mouse—Foot pad
  • Nine-banded armadillo.
 
CANDIDA ALBICANS
Q.1. Identify and describe (Figs 12A and B).
Ans.
  1. Slide of Candida—showing gram positive yeast cells showing budding
  2. Slide of Candida showing pseudohyphae.
zoom view
Figures 12A and B: (A) Yeast cells with budding; (B) Yeast cells with pseudohyphae
Q.2. Mention pathogenicity
Ans.
  • Oral thrush
  • Vulvovaginitis
  • Cutaneous candidiasis
  • 15Paronychia
  • Onychia
  • Intestinal candidiasis
  • Septicemia
  • Endocarditis
  • Meningitis
  • Bronchopulmonary candidiasis
  • Chronic mucocutaneous candidiasis
  • Candida granuloma
  • Urinary tract infection (UTI).
Q3. Mention the medium used for its culture.
Ans.
  • It grows well on Sabouraud's Dextrose agar (SDA)
  • It also grows on bacteriological culture media such as blood agar.
Q.4. Mention the name of the test used for its confirmation.
Ans.
  • Germ tube test (Reynold's Braude phenomenon)
  • Growth on Cornmeal Tween 80 agar. On this agar it forms chlamydospores
  • Carbohydrate assimilation-fermentation tests.
Q.5. Mention the other species.
Ans.
  • Candida stellatoidae
  • Candida tropicalis
  • Candida pseudotropicalis
  • Candida krusei
  • Candida viswanathii
  • Candida guilliermondii
  • Candida parapsilosis.
Q.6. Mention the common sites of the lesions.
Ans.
  • Mouth
  • Vagina
  • Skin
  • Nails
  • Bronchi or lung—less commonly
  • Alimentary tract
  • Heart—less commonly
  • Meninges—less commonly.
 
ACTINOMYCOSIS
Q.1. Identify and describe (Fig. 13).
Ans. Slide of Actinomycosis: This is the slide of sulfur granule obtained from a pus sample obtained from a case of actinomycosis stained with Gram's stain. It shows central gram positive clusters (thin filaments) and peripheral radiating gram negative clubs.
Acid-Fast stains—shows central non-acid fast clubs and peripheral acid fast clubs.
16
zoom view
Figure 13: Actinomycosis
Q.2. Mention the common sites of the lesions.
Ans. Skin, cheek, submaxillary region, lung, abdominal wall, pelvic region.
Q.3. Mention its different species
Ans.
  • Actinomyces israelii
  • Actinomyces bovis
  • Actinomyces viscosus
  • Actinomyces naeslundii
  • Actinomyces odontolyticum
  • Actinomyces gerencsonei.
Q.4. What are the other organisms producing such types of lesions?
Ans.
  • Nocardia asteroids
  • Nocardia brasiliensis
  • Nocardia caviae
  • Actinomadura madurae
  • Streptomyces somaliensis.
Q.5. Mention its treatment.
Ans.
  1. Penicillin or tetracycline for infection due to actinomycetes. Surgery may become necessary if medical treatment fails.
  2. Cotrimoxazole, minocycline, amikacin and cefotaxime for Nocardia.
 
RHINOSPORIDIOSIS
Q.1. Identify and describe (Fig. 14).
17Ans. Slide of Rhinosporidiosis: This is a slide showing larger number of endospores within sporangia embedded in a stroma of connective tissue and capillaries. The sporangium (10–200 mm) contains thousands of endospores (6–7 mm in diameter). These spores when released develop into new sporangia.
zoom view
Figure 14: Rhinosporidiosis
Q.2. Mention the common sites of the lesions.
Ans.
  • Nose
  • Mouth
  • Eye.
Q.3. Mention the causative agent.
Ans. Rhinosporidium seeberi.
Q.4. How is it cultured?
Ans. It is not cultivable on culture media; however, it can be cultured on epithelial cell culture.
Q.5. Where is it common?
Ans.
  • India
  • Shri Lanka.
Q.6. What is the mode of infection?
Ans. Most probably frequent contact with stagnant water or aquatic life.
 
ASPERGILLUS
Q.1. Identify and describe (Fig. 15).
Ans. Slide of Aspergillus (LCB mount): Shows branching and septate hyphae. From the later arise conidiophores, the end of which is expanded to form a vesicle. The vesicle bears one or two layers of sterigmata which in turn bear conidia.
18
zoom view
Figure 15: Aspergillus
Q.2. Mention its pathogenicity.
Ans.
  1. Respiratory:
    • Allergic bronchopulmonary aspergillosis
    • Aspergilloma
    • Invasive aspergillosis
    • Aspergillus asthma.
  2. Superficial infections:
    • Sinusitis
    • Mycotic keratitis
    • Otomycosis.
Q.3. Mention the type of fungus.
Ans. It is a mold.
Q.4. Mention the medium used for culture.
Ans.
  • Sabouraud's Dextrose Agar (SDA) without cycloheximide
  • Brain-heart infusion (BHI).
 
MOLLUSCUM CONTAGIOSUM (FIGS 16A AND B)
Q.1. Identify and describe.
Ans. Slide of Molluscum contagiosum: Slide shows very large (20–30 µ) intracytoplasmic hyaline acidophilic inclusion bodies (Molluscum bodies which displace the nuclei to the periphery) within the proliferated epithelial cells. The molluscum bodies are composed of large number of virus particles.
19
zoom view
Figures 16A and B: Molluscum contagiosum
Q.2. Mention its family.
Ans. Poxviridae.
Q.3. Describe the lesion.
Ans. Pink or pearly white wart like nodules on the skin.
Q.4. Mention its type.
Ans. It is DNA virus of pox group.
Q.5. How is this infection transmitted?
Ans.
  • By direct contact
  • Sexually—in adults.
20
 
MALARIAL PARASITE: PLASMODIUM VIVAX (RING FORM)
Q.1. Identify (Fig. 17). What are they and why?
Ans. These are the ring forms of P. vivax because this is a peripheral blood smear showing ring forms (size 2.5 µ approximately) in the RBCs. The cytoplasm opposite to the nucleus is thick. Nucleus is red and cytoplasm appears blue. There is only one ring per RBC and it is located inside the RBC.
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Figure 17: Plasmodium vivax—ring forms
Q.2. Identify (Fig. 18). What are they and why?
Ans. P. vivax (male gametocyte): This is a slide showing a male gametocyte of Plasmodium vivax. It is approximately 9–10 µ in size. The infected RBC is enlarged. The nucleus is in the center, diffused with malarial pigment dispersed all over the cytoplasm.
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Figure 18: Plasmodium vivax—male gametocyte
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Q.3. Identify (Fig. 19). What are they and why?
Ans. P. vivax (female gametocyte): This is a slide showing the female gametocyte of Plasmodium vivax. It is 10–12 m in size approximately spherical. The nucleus is at the periphery small, compact and surrounded by malarial pigment. The infected RBC is enlarged.
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Figure 19: Plasmodium vivax—female gametocyte
Q.4. Mention the different stains used for staining.
Ans.
  • Leishman stain
  • Giemsa stain
  • Fields stain
  • JSB (J Singh and Bhattacharji) stain
  • Wright's stain.
Q.5. Mention the definitive and intermediate host.
Ans.
  • Definitive—Mosquito (Female Anopheles mosquito)
  • Intermediate—Man.
Q.6. Mention morphological forms of the parasite seen in RBCs.
Ans.
  1. Trophozoite
    • Early—Ring form
    • Late—Ameboid form
  2. Schizont and merozoites
  3. Gametocytes
    • Micro gametocytes (Male gametocytes)
    • Macro gametocytes (Female gametocytes).
Q.7. Mention its media for cultivation. Is culture useful for diagnosis?
Ans. RPMI—1640 medium is used for its culture. Culture is not useful for diagnosis. It is useful for study of its antigenic structure, vaccine preparation and drug sensitivity testing.
Q.8. Mention its pathogenicity.
Ans. Benign tertian malaria.
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PLASMODIUM FACIPARUM (RING FORM)
Q.1. Identify (Figs 20A and B). What are they and why?
Ans. These are the ring forms of P. falciparum because this is a peripheral blood smear showing ring forms (size approximately 1.2–1.5 µ) in the RBCs. The cytoplasm is uniformly thin. There are more than one ring forms in the RBCs. Ring forms are found located inside the RBCs as well as in the surface of the RBCs.
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Figures 20A and B: Plasmodium falciparum—ring forms
Q.2. Identify (Fig. 21). What are they and why?
Ans. P. falciparum (male gametocyte): This is a slide showing the male gametocyte of Plasmodium falciparum. It is 8–10 µ by 2–3 µ and sausage shaped. It is smaller than the female gametocyte and the ends are blunt. The nucleus is central, diffuse with malarial pigment dispersed all over the cytoplasm. The infected RBC is deformed with its membrane stretched over the gametocyte.
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Figure 21: Plasmodium falciparum—male gametocyte
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Q.3. Identify (Figs 22A and 22B). What are they and why?
Ans. Falciparum (female gametocyte): This is a slide showing female gametocyte of Plasmodium falciparum. It is 10–12µ by 2–3µ in size and crescent shaped. The ends of the female gametocyte are pointed. The nucleus is central, compact, surrounded by malarial pigment. The infected RBC is deformed with its membrane stretched over the gametocyte.
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Figure 22A: P. falciparum—female gametocyte
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Figure 22B: P. falciparum—gametocyte
Q.4. Mention the different stains.
Ans. As given for P. vivax.
Q.5. Mention the definite and intermediate host.
Ans. As given for P. vivax.
Q.6. Mention other morphological forms.
Ans. As given for P. vivax.
Q.7. Mention the medium used for its culture.
Ans. As given for P. vivax.
Q.8. Mention its pathogenicity.
Ans.
  • It causes malignant tertian malaria
  • Cerebral malaria
  • Algid malaria
  • Black water fever.
 
ECHINOCOCCUS GRANULOSUS (ADULT FORM)
Q.1. Identify (Figs 23 and 24) and describe.
Ans. Slide of Echinococcus granulosus (adult form): It is 3–6 mm in length, has a scolex and only three segments—the immature, the mature and the gravid segment. The scolex bears four suckers and rostellum with two rows of hooks.
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Figure 23: Echinococcus granulosus—adult
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Figure 24: E. granulosus—scolex
Q.2. Mention the definitive and intermediate host.
Ans.
  • Definitive host—dog, fox, jackal
  • Intermediate—sheep, pig, horse, cattle and man.
Q.3. Mention the diseases produced by it.
Ans. Hydatid disease.
Q.4. Mention the skin test for laboratory diagnosis.
Ans. Casoni's test.
Q.5. Identify (Figs 25 and 26) and describe.
Ans. Slide of hydatid cyst.
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Figure 25: Hydatid sand
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Figure 26: Hydatid cyst
The hydatid cyst is a cyst with the following parts:
  1. The outer layer—which is hyaline, non-nucleated called as ectocyst.
  2. On the inner surface of ectocyst is a layer 10–20 m thick and made of nucleated cells called as endocyst. This is the germinal layer that produces ectocyst, hydatid fluid and brood capsules.
  3. 25Brood capsule is a vesicular structure arising from the endocyst. Several protoscolices develop from the brood capsule. Each protoscolex can give rise to an adult worm.
  4. The broad capsules may break off and settle at the bottom of the cyst. It may also rupture releasing protscolices in the hydatid fluid. The settled brood capsules and protoscolices constitute the hydatid sand.
 
ANCYLOSTOMA DUODENALE
Q.1. Identify (Figs 27 and 28) and describe.
Ans. Ancylostoma duodenale/Hookworm: It is a small grayish white cylindrical worm, male—8 mm and female slightly longer—12.5 mm. The anterior end of the worm is bent slightly dorsally, hence the name hookworm. The posterior end of the male is expanded in an umbrella like fashion, whereas the posterior end of the female is tapering.
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Figures 27A and B: Ancylostoma duodenale
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Figures 28A and B: Ancylostoma duodenale
Q.2. Identify (Figs 29A and B) and describe.
Ans. Egg (Hookworm): The eggs are oval or elliptical 65 µ in length by 40 µ in breath. It is colorless (not bile stained) and surrounded by a transparent hyaline shell membrane. It contains a segmented ovum with four blastomeres, has a clear space between the egg-shell and the segmented ovum.
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Figure 29A and B: (A) Egg of Ancylostoma duodenale; (B) Egg of A. duodenale with 4 blastomeres
Q.3. Mention its pathogenicity.
Ans.
  1. Lesions in skin
    • Ancylostoma dermatitis
    • Creeping eruptions due to larva migrans.
  2. 27Lesions in lung
    • Bronchitis
    • Bronchopneumonia.
  3. Severe anemia (Microcytic hypochromic type)
    • Iron deficiency anemia.
Q.4 Mention the definitive host.
Ans.
  • Man
  • No intermediate host.
 
ENTEROBIUS VERMICULARIS
Q.1. Identify (Figs 30A and 30B) and describe.
Ans. Enterobius vermicularis (Diagram): Thread worm/s (Fig. 30B)—worm/pin worm. It is a small worm white in color, male measures 2–4 mm, and female 8–12 mm. It is more or less spindle shaped.
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Figure 30A: Enterobius vermicularis—adult
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Figure 30B: Photograph of threadworm
Q.2. Identify (Fig. 31) and describe.
Ans. Egg (Enterobius vermicularis): Threadworm—It is an egg which is plano-convex in shape, measures 50–60 m in length by 30 m in breadth. The egg is colorless (not bile stained). It is surrounded by a transparent shell and contains a tadpole-like larva.
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Figure 31: Enterobius vermicularis—egg
Q.3. Mention pathogenicity.
Ans.
  • Enterobiosis
    • Perianal pruritis
    • Nocturnal enuresis
    • May invade female genital tract leading to vulvovaginitis, salpingitis, pelvic or peritoneal granulomas
  • Rarely appendicitis.
Q.4. Mention the definitive host and intermediate host.
Ans.
  • Man
  • No intermediate host.
 
MICROFILARIA
Q.1. Identify (Figs 32 and 33).
Ans. Wuchereria bancrofti—microfilaria.
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Figure 32: Wuchereria bancrofti—microfilaria
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Figure 33: Wuchereria bancrofti—schematic diagram
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Q.2. Mention pathogenicity
Ans.
  • Lymphadenitis
  • Lymphangitis
  • Filarial fever
  • Lymphangiovarix
  • Lymphorrhagia
  • Hydrocele
  • Lymphedema
  • Elephantiasis
  • Occult filariasis
  • Chyluria
  • Chylothorax
  • Chylous ascitis
  • Chylous diarrhea.
Q.3. Describe the provocative test.
Diethyl Carbamazine (DEC), i.e. Hetrazan (100 mg) is given orally. This induces microfilaria to come into peripheral circulation and make its detection easy in peripheral blood smear in day time also. Blood samples are collected 20 to 50 minutes later.
Q.4. Mention the biological vector involved in transmission.
Ans. Mosquitoes: Culex quinquefasciatus (Culex fatigans)—In India and other parts of Asia.
 
RAT FLEA (XENOPSYLLA CHEOPIS)
Q.1. Identify (Fig. 34).
Ans. This is a small wingless insect brown in color with its body compressed laterally and it has long hind legs. So this is Rat flea.
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Figure 34: Rat flea
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Q.2. Mention three diseases transmitted by it.
Ans.
  • Endemic typhus
  • Plague
  • Hymenolepiasis.
 
BODY LOUSE
Q.1. Identify (Fig. 35).
Ans. This is an insect with a small head which has a pair of short antennae. Its thorax is almost square with three pairs of legs. Its abdomen has 9 segments, and the whole insect is flattened dorsoventrally. It does not have wings. Therefore, it is a body louse.
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Figures 35A and B: Body louse
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Q.2. Mention two diseases transmitted by it.
Ans.
  • Epidemic typhus
  • Relapsing fever
  • Trench fever.
 
MITE
Q.1. Identify (Fig. 36).
Ans. This is a small spiderlike (0.55 mm). It is mite.
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Figure 36: Mite
Q.2. Mention three diseases transmitted by it.
Ans.
  • Scabies
  • Scrub typhus
  • Rickettsial pox.
 
TICK
Q.1. Identify (Fig. 37).
Ans. Ticks (soft ticks): The adult tick is 5 mm in length, oval, having four pairs of short legs and when viewed from above, the head is not visible.
Hard tick: This is also oval and has a hard cutaneous shield called scutum which covers the dorsum of hard ticks. They have four pairs of legs and when viewed from above the head is visible. They are dark or brightly colored.
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Figure 37: Tick
Q.2. Mention any two diseases transmitted by it.
Ans.
  1. Tick typhus fevers—Spotted fever
    • Rocky mountain spotted fever
    • Siberian tick typhus
    • South African tick typhus
    • Kenyan tick typhus
    • Indian tick typhus
    • Queensland tick typhus
    • Q fever
  2. Tularemia
  3. Relapsing fever
  4. Arboviral infections
    • Viral encephalitis
    • Kyasanur forest disease
    • Omsk hemorrhagic fever
  5. Lyme disease
  6. Human babesiosis.