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Group: Laboratory Medicine

Subject: General Microbiology and Parasitology (Paper-II)

Q-1-a) Name the disease produced by staphylococcus. (Jan-2022,21)

Ans: Disease/lesions produced by staphylococcus:

CauseSystem involvedLesions

1.Direct invasion or Inflammatory

a. Skin disease
Impetigo, furuncles, carbuncles, paronychia, cellulitis, folliculitis and hydradenitis suppurativa, surgical wound infections, eyelid infections (blepharitis), postpartum breast infections (mastitis).

 b. Septicemia (sepsis)
Sepsis can originate from any localized lesion, specially wound infection, or as a result of intravenous drug abuse.

c. Heart
Infective endocarditis on normal or prosthetic heart valves.

d. Bones and joints
Osteomyelitis (in children) – Septic arthritis

e. Lung
Pneumonia in post-operative patients or following viral respiratory infection.
f. CNSMeningitis, Brain abscess

2. Toxin mediated
– Food poisoning (Gastroenteritis) [Caused by Enterotoxin]Toxic shock syndrome [Caused by Toxic shock syndrome toxin]Scalded skin syndrome [Caused by Exfoliatin]
Q-1-b) Classify bacteria with example. (Jan-2022)

Classification of bacteria:

A. Morphological classification:

1. Cocci: they are rounded. On the basis of their arrangement they are further classified into-

i) Monococcus: They remain single.

ii) Diplococcus: They remain double.

iii) Streptococcus: They remain in chain form.

iv) Staphylococcus: They remain in cluster.

2. Bacilli: They are elongated rod like bacteria, e.g. E. coli.

3. Spirochaetes: They are spiral in shape, e.g. Treponema pallidum.

4. Vibrio: They are comma-shaped, e.g. Vibrio cholerae.

B. On the basis of Gram stain:

1. Gram positive: They are violet in colour on Gram stain. 

2. Gram negative: They are red in colour on Gram stain.

C. On the basis of acid fast staining:

1. Acid fast: e.g. Mycobacterium tuberculosis. 

2. Non-acid fast: e.g. Streptococcus pyogenes, E. coli & most of the bacteria.

D. On the basis of spore formation: 

1. Spore forming bacteria: e.g. Clostridium species, Bacillus species etc.

 2. Non-spore forming bacteria: e.g. Streptococcus pyogenes, Staphylococcus aureus and most of the bacteria.

E. On the basis of presence of cell wall:

1. Bacteria having cell wall: e.g. most of the bacteria.

2. Bacteria lacking cell wall: e.g. mycoplasma.

 Q-1-c) Write down the difference between fungus and bacteria. (Jan-2022)


Difference between fungus and bacteria:

Cell structureProkaryoteEukaryote
Cell wallPeptidoglycanChitin
Other organellesFew or noneMany
NutritionAutotrophs or heterotrophsHeterotrophs
ReproductionSexual and asexualAsexual, sometimes sexual
MovementFlagella or piliNon-motile
HabitatSoil, water, human bodySoil, water, plants, animals
Q-1-d) Name some superficial fungal infections with example. (Jan-2022)

Ans: Superficial Fungal infections of disease with fungal agents:

Fungal infections of diseaseSuperficial fungal agents
Pityriasis versicolor/Tinea versicolorMalassezia furfur
Tinea nigraExophiala werneckii
Black piedraPiedriaea hortae
White piedraTrichosporon cutaneum
Q-1-e) What are the differences between innate and acquired immunity. (Jan-2022)

Ans: Differences between innate and acquired immunity:

TraitsInnate (non-specific) immunityAcquired (specific) immunity
1.Time of developmentPresent form birthAcquired after birth upon contact with antigens
2. SpecificityNon-specificSpecific
3. Response to subsequent exposure of antigenInitial and subsequent responses are samePrimary and secondary responses differ qualitatively and quantitatively
4. Immunological memoryNo Immunological memoryThere is always Immunological memory
5. Present inBoth vertebrates and invertebratesOnly in vertebrates
6. Mechanical barrierPresent (e.g-skin & mucousmembrane).Absent
7. Cells involvedMacrophages,polymorphonuclear leukocytes,natural killer cellsPredominantly T and B-lymphocytes
Q-2-a) Write down the safety code of practices for microbiology laboratory. (Jan-2022)


Code of Safe Laboratory Practice :- 

1. Mouth pipetting or specimens should be avoided.

2. Keeping the laboratory clean.

3. Work spaces should be decontaminated everyday.

4. Washing and sterilization of reusable specimens. containers, needles, syringes, lancets,    slides, cover, pipettes  by appropriate procedures.

5. Hand should be washed by using chemical disinfectants such as 5%, Phenol or Lysol, after handling infectious material.

6. Wearing an apron and safe footwear, use of protective gloves, goggles, face shield, dust  mask, eyewash bottle.

7. Laboratory doors should be kept closed when work is in progress.

8. Eating, drinking, storing food and smoking should not be permitted in the laboratory.

9. Control of noise and other causes of loss of concentration.

10. Proper maintenance and routine cleaning of equipment.

11. Safe handling and storage of chemicals and reagents.

12. Regulations covering the safe packing and transport of specimens.

Q-2-b) What are the types of microscope? Write about care of microscope. (Jan-2022)


Types of microscope:

1.Simple microscope.

2.Compound/light microscope.

There are many types of light microscope,such as-

  1. Bright field microscope
  2. Dark field microscope
  3. Phase contrast microscope
  4. Polarizing microscope
  5. Fluorescence microscope

3.Electron microscope

a)Transmission electron microscope

b)Scanning electron microscope

Care of microscope :

1. Examine all the parts of a microscope before use.

2. Keep the microscope in a perfectly clean condition.

3. Carry by holding the arm.

4. Clean the lenses with a Soft Cotton Cloth moistened with xylol. Remove xylol with a dry cloth without delay.

5. Clean the oil-immersion objective on each occasion after use, by wiping the oil from the front of the Lens with a cloth.

Q-2-c) Draw and label a bacterial spore. Name some spore bearing organisms. (Jan-2022)


Bacterial Spore
  Spore bearing organism:
Bacillus anthracis
Bacillus subtilis
Clostridium Tetani
Clostridium perfringens
Clostridium Botulinum
Q-2-d) Name the malarial parasite.Give the laboratory diagnosis of malaria. (Jan-2022)

Malarial parasites:

  • Plasmodium vivax
  • Plasmodium falciparum 
  • Plasmodium ovale
  •  Plasmodium malariae

Laboratory diagnosis of malaria:


Diagnosis of malaria depends on demonstration of malarial parasite in the peripheral blood film, detection of antibody or antigen from blood, and also by nucleic acid based techniques.


1) Specimen collection: Blood.

2) Microscopic examination: Leishman (or Giemsa) stained smear of thick and thin blood films.

  •  Thick smear – to detect the presence of parasite.
  • Thin smear-to detect the species of parasite.

3) Culture: rarely done where there arrives difficulties in detecting the parasite.

4) Antigen detection: Immunochromatographic ‘dipstick’ tests, also called rapid diagnostic tests (RDTs).

5) Serological tests: Does not detect current infection, but rather measures past experience. 

  • IFAT-for detection of antibody in chronic malaria.
  • ELISA.

6) Blood count: Leucopenia with monocytosis (15-20%)

7) Nucleic acid based technique: PCR. (Polymerase Chain Reaction)

Q-2-e) Write down the difference between gram positive and gram negative bacteria. (Jan-2022)

Ans: Difference between gram positive and gram negative bacteria:

ComponentGram positive bacteriaGram negative bacteria
1. PeptidoglycanThicker, may be 40 layersThinner, 1-2 Layers
2. Teichoic acidPresentAbsent
3. Lipopolysaccharide (endotoxin)AbsentPresent
4. Outer membraneAbsentPresent
5. Periplasmic spaceAbsentPresent
6. Gram stainBlue (violet)Red
Q-3-a) Write down the basic steps in a staining procedure. Describe the steps of Ziehi Neelsen’s staining procedure. (Jan-2022).


 Basic steps in a staining procedure:

  1. Preparation and fixation of the film 
  2. Staining 
  3. Decolourisation 
  4. Counter Stain 
  5. Wash and dry 
  6. Result

Acid Fast Bacillus (AFB) e.g-mycobacterium tuberculosis is stained by Ziehl-Neelsen (ZN) staining.


  • Glass slide
  • Cotton
  • Platinum loop/wire loop
  • Test tube with holder
  • Staining rack 
  • Spirit lamp 
  • Match
  • Microscope


  • Carbol fuchsin Solution
  • 20%  H2So4
  • Methylene blue / Malachite green
  • Distilled water 
  • cedar wood oil.

Specimen: Sputum


1. clean the slide and make it greeze-free.

2. Make a thin, uniform film with a sterilized loop.

3. Dry the film in air.

 4. Fix the film by slowly passing the slide 3-4 times through a flame.

5. Heat carbol fuchsin in a test tube till fumes appear.

6. Cover the slide with fuming carbol fuchsin and Keep for 5 minutes.

7. wash with water.

8. Decolourize with 20% sulphuric acid or acid alcohol until only a faint pink colour remains.

9. wash with water. 

10. Counterstain with methylene blue for 20-30 seconds.

11. wash with water and dry in the air.

Examine: Examine under oil-immersion of the microscope.

Q-3-b) Define parasite and host. What are the relations between host and parasite. (Jan-2022)


Parasite can be defined as a living organism which receives nourishment and shelter from another organism where it lives.


Hosts are organisms which gives shelter and nourishment to the parasite.

Host-parasite relationship:

Host-parasite relationship/Types of biological association:

 An association, which is formed between animals of different species, may be divided into 3 categories –


An association in which both are so dependent upon each other that one cannot live without the help of others. None of the partners suffer harm from the association. 

Example: Bacterial flora in the human intestine.


An association in which the parasite only is deriving nutrition without causing harm to the host. A commensal is capable of leading independent life.

 Example: E. coli in the human intestine.


An association in which the parasite derives benefit and the host gets nothing in return but always suffers from injury.

 Example: Man & hook worm.

Q-3-c) Define and classify virus with example. (Jan-2022)


Viruses are particles composed of an internal core containing either DNA or RNA (but not both) covered by a protective protein coat.

Classification of Viruses:

1) On the basis of type of nucleic acid:

a) DNA viruses

  • Poxvirus. 
  •  Herpes virus.

b) RNA viruses

  • Corona virus
  • Reo virus

2) On the basis of strandedness:

a) Double stranded viruses

  • Hepatitis B virus
  • Rota virus
  • Smallpox virus

b) Single stranded virus

  • Dengue virus
  • Polio virus
  • Messles virus

3) On the basis of presence of envelop:

a) Enveloped viruses

  • Hepatitis B virus 
  • Small-pox virus

b) Non-enveloped viruses

  • Hepatitis A virus
  • Rota virus

4) On the basis of character of nucleic acid:

a) Positive strand viruses

  • Hepatitis A virus
  • Polio virus
  • Dengue virus

b) Negative strand viruses

  • Influenza virus
  • Measles virus

5) On the basis of symmetry of genome:

a) Icosahedral capsid:

  • Hepatitis B virus
  • Herpes simplex virus

b) Helical capsid:

  • Influenza virus
  • Measles virus
  • Rabies virus

6) On the basis of ether sensitivity:

a) Ether sensitive:

  • Herpes simplex virus
  • Corona virus

b) Ether resistant:

  • Pox virus
  • Adenovirus 
Q-3-d) Define bacterial growth curve.Describe the utility bacterial growth curve. (Jan-2022)

Bacterial growth curve : 

If a small number of bacteria is inoculated into a liquid nutrient medium and bacteria is counted of  frequent intervals and a graphical demonstration is produced by putting log number of cells along the Y-axis and time along the X-axis, then it is known as bacterial growth curve.

Utility of bacterial growth curve:

A bacterial growth curve is a graph that shows the number of bacteria in a culture over time. It is typically divided into four phases:

  • Lag phase: This is the initial phase of growth, where the bacteria are adapting to the new environment. The number of bacteria does not change significantly during this phase.
  • Log phase: This is the phase of exponential growth, where the number of bacteria doubles every few minutes. This is the fastest phase of growth.
  • Stationary phase: This is the phase where the growth of the bacteria slows down as the nutrients in the environment become depleted. The number of bacteria may stay constant or even start to decline.
  • Death phase: This is the phase where the number of bacteria declines rapidly as they die off due to lack of nutrients or the accumulation of toxic waste products.
Q-3-e) Define and classify culture media with example. (Jan-2022)

Media/Culture media:

Media may be defined as an artificial food for the cultivation of bacteria in the laboratory.

Classification of media:

A. On the basis of consistency:

1) Solid media: e.g. nutrient agar, blood agar, chocolate agar etc .

2) Semisolid media: e.g. soft agar media.

3) Liquid media: Peptone water, bile broth, sugar broth, Loeffler’s serum, Robertson’s cooked meat media etc.

B. On the basis of chemical/nutritional composition:

1) Simple/basal media: For general purpose. Example – nutrient broth, nutrient agar, peptone water, glucose broth etc.

2) Special media:

i) Enriched media: e.g. blood agar medium, Lowenstein-Jensen medium.

 ii) Enrichment media: e.g. Tryptic soya broth.

iii) Selective media: e.g. Mac Conkey’s agar, blood tellurite agar.

iv) Indicator/ differential media: e.g. blood agar, Mac Conkey’s agar.

 v) Transport media: e.g. Stuart medium, Carry-Blair medium.

vi) Storage media: e.g. egg-saline medium.

C. On the basis of oxygen requirement:

  • Aerobic media.
  • Anaerobic media.
Q-4-a) Write down the EPI immunization schedule in Bangladesh. (Jan-2022)

EPI immunization schedule in Bangladesh:

Name of diseaseName of vaccineQuantity for each doseInterval between doseNumber of DoseTime of startingTime of completionSite for vaccinationRoute of administration
TuberculosisBCG0.05ml1After birth1 yearUpper part of the left armIntradermal
Diphtheria, Pertussis, Tetanus, Hepatitis -B, PneumoniaPentavalent0.5ml28 days342days after birth1 yearThighIntramuscular
Polio-myelitisOPV2 drops28 days442 days after birth1 yearMouthBy dropper
TetanusTT0.5ml15 years =first. After 28 days=2nd. After 6 month=3rd. After 1 year=4th. After 1 year=5th.515 year15-49 year Upper part of both armIntramuscular
Measles RubellaMR0.5ml19 month18monthThigh (Right)Subcutaneous 
Q-4-b) Describe the collection and transport of microbiological specimen. (Jan-2022)


The collection and transport of microbiological specimens is an important step in the diagnosis and treatment of infectious diseases. The following are some general guidelines for collecting and transporting microbiological specimens:

  • The specimen should be collected as soon as possible after the onset of symptoms.
  • The specimen should be collected from a sterile site, if possible.
  • The specimen should be collected in a clean, dry container.
  • The container should be labeled with the patient’s name, identification number, date and time of collection, and the source of the specimen.
  • The specimen should be transported to the laboratory as soon as possible.
  • If the specimen cannot be transported immediately, it should be refrigerated.
Q-4-c) Write in short about bacterial pathogenicity and virulence. (Jan-2022)


Bacterial pathogenicity is the ability of a bacterium to cause disease. Virulence is the degree of pathogenicity of a bacterium, or how likely it is to cause disease.

There are many factors that contribute to bacterial pathogenicity and virulence, including:

  • The type of bacterium: Some bacteria are more pathogenic than others. For example, Escherichia coli is a common bacterium that is usually harmless, but some strains of E. coli can cause food poisoning.
  • The number of bacteria: The more bacteria that are present, the more likely they are to cause disease.
  • The route of infection: The way that bacteria enter the body can also affect their pathogenicity. For example, bacteria that enter the bloodstream are more likely to cause serious disease than bacteria that enter the skin.
  • The host’s immune system: The health of the host’s immune system also plays a role in bacterial pathogenicity. People with weakened immune systems are more likely to get sick from bacteria.

Bacterial virulence factors are the characteristics that contribute to a bacterium’s pathogenicity. They can be divided into three main categories:

  • Adhesins: These are molecules that allow bacteria to attach to host cells.
  • Invasins: These are molecules that allow bacteria to enter and invade host cells.
  • Toxins: These are poisonous substances that can damage host cells.
Q-4-d) Name the blood borne parasites. Write down the laboratory diagnosis of kala-azar. (Jan-2022)

Blood borne parasite :

The blood borne parasites which can be found in the bloodstream of an infected person are called blood borne parasites. 

Name some blood borne parasite :

  • Leishmania donovani causes Kala-azar
  • Plasmodium species causes Malaria
  • Wuchereria bancrofti causes Filariasis 

Laboratory diagnosis of kala-azar:


 Leishmaniasis can be diagnosed by demonstration of the parasite (LD bodies) from the cutaneous, mucosal lesions and incase of visceral leishmaniasis from Bone marrow, Spleen or Lymph node aspirate. It can also be diagnosed by detection of antigen or antibody by immunological tests and isolation of the organism by culture if facilities are available. Nucleic acid techniques are also helpful.

Specimen collection:

 Aspiration of bone marrow, spleen, lymph nodes, buffy coat of blood.

Microscopic examinations:

  • Leishman (or Giemsa) stained smear.
  •  Findings: Amastigote form (LD bodies) in bone marrow, splenic aspirate and within blood monocytes.

Isolation and identification from culture:

  •  NNN media (Novy, Mac Neal, Nicole).
  • Incubation- at 22° C.
  • Incubation time-1 to 2 weeks.
  • Findings- promastigote forms of parasites are found.

Serological tests:


For detection of antibody

  • ICT (ImmunoChromatographic Test)
  • DAT (Direct Agglutination Test)
  • IFAT (Indirect Fluorescent Antibody Test)
  • CFT (Complement Fixation Test)

For detection of antigen-

  • ELISA (Enzyme Linked ImmunoSorbent Assay)
  •  RIA (Radio Immuno Assay)


  • Aldehyde test for detection of high globulin.
  • Antimony test.
  • Blood count-Decrease WBC. RBC, Platelet.

NA based techniques: 

PCR (Polymerase chain reaction).

Q-4-e) Write down the steps of Gram’s stain. (Jan-2022) 

Definition: Gram staining is the commonest  staining  method used in the clinical bacteriology. which divides most of the bacteria into two major groups.


  1. Glass slide
  2. Cotton
  3. Platinum loop/Wire loop
  4. Spirit Lamp
  5. Culture media
  6. Staining rack
  7.  Microscope


  1. 0.5% crystal violet or gentian violet or methylene violet
  2. Lugol’s iodine
  3. Alcohol / Acetone.
  4. Distilled water.
  5. 1 in 10 dilute carbol fuchsin solution.
  6. Cedar wood oil
  7. Normal saline

Specimen : Bacterial culture colony (Pus, Body fluid, Sputum)


1. Clean the slide and make it greeze free.

2. Make a thin, uniform film with a Stenitized loop. 

3. Dry the film in air.

4. Fix the film by slowly passing the slide 3-4 times through a flame.

5. Cover the film with gention on crystal violet stain. Keep for 2 to 5 mins.

6. Drain off the stain.

7. Cover with Gram’s iodine for 1 to 2 minutes (mordanting).

8. Decolorize with acetone or alcohol by gentle agitation and then wash with water. Repeat the alternate washing with alcohol and water until the colour ceases to come out of the film. Avoid further washing.

9. Cover with 1 in 10 dilute carbol fuchsin 20 to 30 seconds.

10.wash with water and dry in air.

Examination: Examine under oil immersion of the microscope.

Q-5-a) Write  short note on: Tuberculin test. (Jan-2022)

Tuberculin Test (Mantoux test):

It is a hypersensitivity test that depends on delayed hypersensitivity reaction, which develops in individuals.

Purpose (use) of the test:

i) Aid to diagnosis. 

ii) For contact tracing.

iii) For post-reaction check up.


Tuberculin reagent is a purified protein derivative (PPD) of mycobacteria. ‘WHO-recommended tuberculin’ is “2TU PPD Rt 23′ ‘ diluted by Tueen80.

Technique of injection:

0.1 ml volume of tuberculin is slowly injected on the flexor aspect of the fore-arm intradermally.

Measurement of reaction:

The test is examined after 3 days and is carefully palpated for any induration.

Interpretation of the test:


  • If induration is 10 mm or more: Positive (+ve).
  • If induration is less than 10 mm: Negative (-ve)

Limitation of tuberculin test: 

          A. Tuberculin test may be positive other than active tuberculosis in-

                                                   i. Past TB

                                                   ii. Previous vaccination.

           B.Tuberculin test may be negative in spite of having TB (false negative) in-

  • Severe tuberculosis (25% of cases negative) 
  •  Extremes of age (newborn & old age)
  •  Malnutrition
  • HIV (if CD4 count <200 cells/ml)
  • Immunosuppressive drugs
  • Malignancy
  • Sarcoidosis etc.
Q-5-b) Write short note on: Hydatid cyst. (Jan-2022)

Hydatid cyst:

Definition: The larval worm of E. granulosus in man causes unilocular hydatid disease.

Laboratory diagnosis:


The diagnosis of Echinococcosis relies mainly on findings by ultrasonography and/or other imaging techniques supported by positive serologic tests for detection of antibody. Microscopy can be done but there is risk of hypersensitivity and dissemination to other organs.

Specimen collection:

  • Blood.
  •  Aspirated fluid.

Microscopic examination:

  • Examine the explorated fluid for scolices or hooklets.

Blood examination:

  • Eosinophilia (20 to 25%)

Serological tests:

  • Indirect haemagglutination test, IFAT.

Casoni’s test:

  • Specific and widely practiced.
Q-5-c) Write short note on: Pasteurization. (Jan-2022)


Pasteurization may be defined as the process by which milk is heated upto such a temperature & for such a period of time to destroy the microorganisms within the milk without altering the composition, flavor color & nutritive value of milk.


1. Holding method – 63°C for 30 minutes

2. Flash method-72°C for 15 seconds.

3. HTST method – High temperature for a short time.


The sterilization/killing of organisms of milk & milk products causes milk borne diseases.

Bacteria removed by pasteurization:

  • Mycobacterium bovis
  • Salmonella
  • Escherichia coli
  • Brucella abortus

Advantages of pasteurization over the boiling:

 Boiling causes destruction of many nutrients & flavour of the milk, but pasteurization preserves all the quality of the milk.

Q-5-d) Write short note on : Flotation technique. (Jan-2022)

Flotation technique:

Flotation technique is a method used to concentrate and isolate parasites from stool samples.


The most common flotation solutions used are zinc sulfate and Sheather’s sugar. Zinc sulfate has a specific gravity of 1.18, which is slightly higher than the specific gravity of most parasites. Sheather’s sugar has a specific gravity of 1.20, which is even higher and can float more parasites.


To perform a flotation technique, a small amount of stool is mixed with the flotation solution in a test tube. The tube is then centrifuged for a few minutes, which helps to sediment the debris and parasites to the bottom of the tube. The supernatant, which contains the parasites, is then carefully pipetted off and examined under a microscope.

Advantages of Flotation technique:

  • Sensitive method for detecting parasites
  • Can be used to detect a wide variety of parasites
  • Relatively inexpensive

Disadvantages of Flotation technique:

  • Time-consuming and labor-intensive
  • Not all parasites will float
  • Can damage parasites
Q-5-e) Write short note on : Chocolate agar media. (Jan-2022)

Chocolate agar media:

Chocolate agar is a non-selective, enriched growth medium used for isolation of pathogenic bacteria. 

Used of Chocolate agar media:

Chocolate agar is used for growing fastidious respiratory bacteria, such as Haemophilus influenzae and Neisseria meningitidis.

Composition of chocolate agar media:

  • Peptone: A source of amino acids and peptides for bacterial growth.
  • Yeast extract: Provides carbohydrates as an energy source for bacteria.
  • Sodium chloride: Maintains osmotic balance in the medium.
  • Dipotassium phosphate: Acts as a pH buffer to maintain the desired pH for bacterial growth.
  • Hemin: A growth factor required by certain bacteria, such as Haemophilus influenzae.
  • NAD: A coenzyme needed for specific metabolic pathways of bacteria.
  • Blood: Provides iron and other nutrients necessary for bacterial growth.

Advantages of chocolate agar media:

  • Enriched with nutrients that are required by fastidious bacteria.
  • Provides CO2, which is required by some bacteria for growth.
  • Easy to prepare and use.

Disadvantages of chocolate agar media:

  • Can be expensive to purchase.
  • Not all bacteria will grow on chocolate agar.
  • Can be contaminated easily.
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