SPOTTERS

1) Bacteriological Loop
1. The loop is flat and circular and completely closed with 2-4 mm internal
diameter
2. The loops are made of Nichrome wire no 24 SWG
Uses:
 Used for inoculating various clinical specimens on solid or liquid media.
 Used in streak culture and stroke culture.
 Used to put up biochemical reactions.

2) Sterile Swab
1. Swab sticks are sterilized by hot air oven
2. Presterilized disposable swabs are commercially prepared and sterilized by
gamma radiation
Uses:
 Collection of specimens from various sites - Nasopharynx, Oropharynx,
etc.
 Preparation of lawn culture of bacterial growth as in antibiotic sensitivity
testing

3) Sterile Disposable syringe

1. Sterile disposable syringes are pre sterilized by either gamma radiation or
ethylene oxide
2. Once used they are discarded and never reused.
Uses:
 For collection of various clinical specimens like blood, sterile body fluids
& pus
 For injecting medications to the patients

4) Inspissator
 It is a Heat based physical method achieving intermediate level disinfection
 Egg based culture media such as LJ medium are sterilized
 Sterilized by heating at 80-85˚C for 30 minutes on 3 successive days so that
spores are killed.

5) Hot air oven

 Mechanism of action & principle is based on protein denaturation and
oxidative damage.
 Called as dry heat sterilizer used to sterilize materials that might be
damaged by moist heat or that are impenetrable to moist heat like
glassware, swabs, paraffin &dusting powder
 It is nontoxic, non-corrosive and penetrate well into materials

6) Nutrient agar plate

 Simple / basal medium made up of nutrient broth and 2% agar
 Preferred medium to study the colony morphology and pigment
demonstration
 They serve as a base for preparation of many other media

7) MacConkey agar plate

 It is a differential and low selective medium
 It differentiates organism into Lactose fermenters (Pink colored colonies
due to lactose fermentation) and non-lactosefermenters (Colorless colonies
due to non-lactosefermentation)
 Contains peptone, lactose, agar, neutral red (indicator)and taurocholate
(PLANT)

8) Blood agar plate

 Enriched media which supports the growth of fastidious organism.
 It is prepared by adding 5-10% of homogenized, defibrinated & sterile
sheep blood to the molten agar at 45˚C
 Tests the hemolytic property of the bacteria which maybe (α hemolysis) or
complete (β hemolysis)

9) Chocolate agar plate

 Enriched medium
 Heated blood agar prepared by adding 5-10% sheep blood to molten
nutrient agar at 70˚C
 Supports growth of highly fastidious organism like Haemophilus influenza

10) TCBS (Thiosulfate Citrate Bile salt Sucrose) Agar plate

 Selective medium for vibrio species
 Contains Thiosulfate, Citrate, Bile salt, Sucrose at pH 8.6
 Bromothymol blue and thymol blue are indicators
 Sabouraud Vibriochloreae produces yellow-colored colonies

11) dextrose agar – slope

• It is the culture media used for isolation of fungi – yeasts and molds.
 • Contains peptone, dextrose 4% and pH 5.6, increased dextrose
concentration and acidic pH enhances growth of fungi and inhibits
bacterial growth.
• Gentamycin / tetracycline / Chloramphenicol can be added to inhibit
the growth of bacteria

12) Robertson’s Cooked Meat medium

• Used for the cultivation of anaerobic bacteria
• Contains chopped meat particles in nutrient broth which provide
glutathione (reducing agent) and unsaturated fatty acid.

13) Robert Koch

• Father of Bacteriology
• Introduced Solid media – agar
• Discovered anthrax bacilli, tubercle bacilli, and cholera bacilli.
• Described hanging drop method for testing motility.
14) Louis Pasteur
• Father of Microbiology
• Proposed the principles of fermentation
• Disproved spontaneous generation theory and proposed germ theory of
disease.
• Described the method of Pasteurization of milk.

15) Triple sugar iron agar- Acid slant/acid butt

• Triple sugar iron agar with Acid slant / acid butt, a composite solid
medium widely used for the identification of Gram-negative bacilli.
• It contains Glucose, Lactose and sucrose in the ratio of 1:10:10 parts
• The organism ferments more than 2 sugars (glucose & also ferments
lactose and (or) sucrose) to produce large amount of acid, so that the
medium turns acidic (yellow) at 8 hours. At 18 – 24 hours, the
medium maintains Acidic pH both in slant and butt and gives an acid
(yellow) slant / acid butt or A/A reaction.
• Eg: Escherichia coli, Klebsiella species
16) Triple sugar iron agar- Alkaline slant/alkaline butt
• TSI with Alkaline slant / Alkaline butt
• It indicates Non fermentation of carbohydrates. Hence the medium
remains red.
• Eg: Pseudomonas species, Acinetobacter species.

17) Triple sugar iron agar- Alkaline slant/acid butt

• TSI with Alkaline slant / Acid butt.
• It indicates fermentation of glucose only & produce little acid only at
the butt whereas slant remains alkaline.
• Eg: Shigella sonnei

18) Triple sugar iron agar- Alkaline slant/acid butt WITH PROFUSE
H2S
• Alkaline slant / Acid butt with Hydrogen sulphide.
• It indicates fermentation of glucose only & produce little acid only at
the butt whereas slant remains alkaline.
• Eg: Proteus vulgaris
19) Triple sugar iron agar- Alkaline slant/acid butt WITH SPECK
OF H2S
• Alkaline slant / Acid butt with speck of H2S
• It indicates fermentation of glucose only & produce little acid only at
the butt whereas slant remains alkaline.
• Eg: Salmonella typhi.

20) Oxidase Test

• It detects the presence of Cytochrome Oxidase enzyme in bacteria, which
catalyses the oxidation of reduced Cytochrome by atmospheric oxygen.
• Oxidase agent is Tetramethyl-p-phenylene diamino dihydrochloride.
• Appearance of purple colour is considered to be positive
• Eg: Oxidase Positive – Pseudomonas,Vibrio, Neisseria
• Oxidase Negative - Enterobacteriaceae.
21) Antibiogram
• An Antibiogram is an overall profile of antimicrobial susceptibility testing
results of a specific microorganism to a battery of antimicrobial agents.
• Specific microorganism is lawn cultured onto Mueller Hinton agar and
antimicrobial discs are kept over it and the zone of inhibition of each
antimicrobial agent is measured after 18 to 24 hours of incubation
• It is also a useful tool for detecting and monitoring trends in antimicrobial
resistance within the hospital.

22) INDOLE TEST – POSITIVE

• A red coloured ring is formed near the surface of the 24hrs young peptone
water broth on addition of Kovac’s reagent is Indole positive.
• It detects the ability of bacteria to produce enzyme tryptophanase that
breakdown amino acid tryptophanase present in the medium into indole.
• Eg:Positive- Escherichia coli, Proteus vulgaris.
• Eg: Negative-Pseudomonas species, Acinetobacter species

23) CITRATE TEST – UTILIZED

CITRATE TEST – NOT UTILIZED
• Citrate utilizing bacteria produce growth and a colour change (i.e., Original
green colour changes to blue colour) in Simmon’s citrate medium.
• Bromothymol blue is the pH indicator.
• Eg: Klebsiella pneumoniae, Citrobacter, Enterobacter.
• Eg: Escherichia coli, Salmonella typhi
24) UREASE TEST – Urea HYDROLYSED
UREASE TEST – Urea NOT HYDROLYSED
• Urease producing bacteria turns the Christensen’s urea medium to pink
colour by splitting urea to ammonia and makes the medium alkaline.
• Phenol red acts as pH indicator
• Eg: Klebsiella pneumoniae, Proteus species, Helicobacter pylori.
• Eg: Escherichia coli, Salmonella typhi

25) COAGULASE TEST

1. It detects of the ability of the organism to produce Coagulase.
2. It differentiates Staphylococcus aureus and CoNS
3. Positive test is indicated by formation of a clot that does not flow when the
test tube is tilted.
4. Eg: Staphylococcus aureus

26) CATALASE TEST

• It detects the ability of certain bacteria which has the enzyme Catalase
which acts on hydrogen peroxide to release oxygen.
• H202Catalase H20 + O (Nascentoxygen )
• Positive test is indicated by immediate bubbling easily absorbed due to O2
formed.
• Examples of Catalase positive organism
• Escherichia coli
• Klebsiella species
• Staphylococcus species

27) Sterile syringe with needle and vacutainer tubes

• Blood sample collection method:
• Label the vacutainer with the patients name,age , sex, IP/OP no, ward no
,and date and time of collection
• Wash hands thoroughly and put on gloves. Tie a tourniquet
• clean the venipuncture area with a bactericidal agent (2% tincture iodine
/70% alcohol/chlorhexidine) beginning at the centre of the site and cleaning
in a circular motion outward to a diameter of three or four inches for about
30 seconds. Donot go back over the previously cleaned areas
• Do not touch the venipuncture area after disinfection. Collect the required
volume of blood with a sterile needle and syringe and inoculate.
Colour Anticoagulant Uses
Red tube No anticoagulant Serological examination
Grey tube Sodium fluoride Glucose examination
Haematological
examination like
Purple tube EDTA
complete hemogram,
ESR
Coagulation studies like
Blue tube 3.2% sodium citrate
PT, APTT
Green tube Heparin Bone marrow studies

28) ELISA – Microtitre plate

• Materials used are Polystyrene an& Polypropylene
• ELISA microplates are ideal for biochemical cell-based assays designed to
identify Quantify protein species
• It contains 96 well plate assay for studying bio film formation, which
allows for the observation of bacterial adherence to an abiotic surface.
• A buffered solution of the antigen to be tested for is added to each well of a
Microtiter plate where it is given time to adhere to the plastic through
charge interactions.

29) IMMUNOCHROMOTOGRAPHY TEST

1. It utilizes the principle of lateral flow or agglutination that forms the
antigen – antibody complexes with the specific antigen of the pathogen
from the given sample. It is sandwich immunoassay; the test uses a
nitrocellulose strip with a conjugate site containing antigens.
2. The sample is taken and added to the well of the test card
3. Due to the capillary action the mixture flows down to the test pad the
antibodies present in the sample binds to the antigen then the formed
conjugate complex passes to the nitrocellulose membrane and comes in
contact with the test lines.
4. Immunochromatography qualitatively detects Hepatitis B surface antigen.

30) NAP with green pigment colonies

• Pseudomonas aeruginosa produces green pigmentation due to
pyocyanin,a diffusible pigment
• Pseudomonas aeruginosa produces large opaque,irregular colonies
with a metallic sheen (described as Iridescence ) in nutrient agar

31) Nutrient agar – Golden yellow pigment

• Staphylococcus aureus produces golden yellow non diffusible
pigment – Staphyloxanthin
• Pigments are made up of ß – Carotene
 • Staphyloxanthin acts as an important virulence factor due to its anti-
oxidant properties.

32) MacConkey agar – lactose fermenting colonies

• Produces pink colour colonies due to lactose fermentation.
• PH indicator- Neutral red
• Eg: Escherichia coli, Klebsiella spp

33) MacConkey agar – non lactose fermenting colonies

• Produces colourless colonies due to non-lactose fermenters.
• PH indicator- Neutral red
• Eg: Salmonella spp, Proteus spp.

34) GPC Clusters

• The given smear shows gram positive spherical cocci, arranged in grape-
like clusters i.e. Staphylococcus aureus
• This arrangement is due to cell division in Staphylococcus aureus occurs in
multiple planes with daughter cells remaining close together.
• Staphylococcus aureus is most virulent species among Staphylococci,
produces infection ranging from localized pyogenic infection to life
threatening systemic infection.

35) GPC in pairs

• The given smear shows gram positive cocci in pairs- S. pneumoniae.
• They are present in upper respiratory tract of human beings as commensals.
• They are the leading cause of lobar pneumonia & otitis media in children
and Meningitis in all ages.

36) GPC in chains

• The given smear shows Gram positive cocci in long chains – Viridans
streptococci.
• They are commensals of mouth & upper respiratory tract.
• Viridans Streptococci are most common cause of SABE. The commensal
Viridians Streptococci (Streptococcus.sanguis) in the oral cavity can enter
blood to cause transient bacteremia while chewing, tooth brushing& dental
procedures that can account for the predilection of those organisms to cause
endocarditis.

37) AFB
• The given smear shows long, slender, beaded &pink-coloredacid-fast
bacilli i.e.
• Mycobacterium tuberculosis, causative agent of TB in human beings.
• Acid fastness is due to the mycolic acid present in the bacterial cell wall.

VIROLOGY

1) BACTERIOPHAGES
1. Viruses that infect bacteria are called bacteriophages
2. They are tadpole shaped with hexagonal head enclosing dsDNA and tail
ending with tail fibers
Uses:
• Phage therapy
• As cloning vector in recombinant DNA technology
 • Transfer drug resistance

2) RABIES VIRUS
1. Bullet shaped virus – belongs to Rhabdoviridae family
2. They have lipid envelope lined internally by a layer of matrix proteins and
externally by long peplomers or spikes
3. Nucleocapsid have a single stranded negative sense RNA, nucleoprotein
and polymerase protein

3) CORONA VIRUS
1. RNA virus which has nucleocapsid with a helical symmetry surrounded by
an envelope
2. It possesses structural proteins such as nucleocapsid protein (N), spike
protein (S), Membrane glycoprotein (M) and envelope protein (E)

4) HIV Virus – Human Immunodeficiency Virus

1. It is a spherical and enveloped virus
2. Envelope is made up of lipid part and protein part (gp 120 &gp 41)
3. Nucleocapsid is icosahedral in symmetry which encloses 2 identical copies
of ssRNA and viral enzymes such as reverse transcriptase ,integrase &
proteases

PARASITOLGY

1) Ascaris male
1. Adult Ascaris worm is elongated, cylindrical and unsegmented.
2. Female worm is longer than male.
3. Posterior end is curved ventrally to form a hook and carries 2 copulatory
spicules

2) Ascaris female
1. Adult Ascaris worm is elongated, cylindrical and unsegmented.
2. Female worm is longer than male.
3. Its posterior extremity is straight and conical
4. Female worm contains vulvar waist or genital girdle to facilitate mating

3) Tape worm
• Tape worms are long, segmented and dorsoventrally flattened worms.
• Adult worm consists of 3 parts – head or scolex, neck and strobili.
• Suckers in scolex help in attachment of worm to intestinal mucosa.

4) Hydatid Cyst
• It is the larval form of Echinococcus granulosus
• It is a fluid filled bladder like structure ,unilocular, subspherical in shape,
with an average size of 5 - 8 cm
• The cyst has three layers- outer pericyst, middle ectocyst and inner
endocyst
• Cyst contains clear,pale, yellow coloured fluid called hydatid fluid.

5) Tape Worm- Proglottids

• Proglottids are segments of the body of tapeworm.
• Based on the reproductive organs they bear, proglottids can further be
grouped into immature, mature and gravid segments
• In Immature segments, male and female reproductive organs are not
differentiated.
• Mature segments contain male and female organs in the same segment
• Gravid segments or fertilized segments are formed after fertilization, the
uterus gets filled with eggs.

6) Fasciola Hepatica
1. The worm is bilaterally symmetrical,unsegmented , leaf like , flattened
dorsoventrally.
2. It has no true body cavity.
3. The worm contains oral and ventral suckers.
4. The intestinal caeca and testes are extensively branched.

MYCOLOGY

1) Aspergillus Niger (Macroscopic)

• Sabourad’s dextrose agar slope with woolly to powdery or granular due to
dense condiation (spore formation)- rapid grower

2) Aspergillus Niger (Microscopic)

• Lactophenol cotton blue mount showing
- Thin, hyaline septate hyphae
- Conidiophore arises from hyphae at an acute angle which ends at
spherical vesicle
- Biseriate phialides
 - Conidia occupies the entire vesicle and are black in colour
3) Aspergillus flavus (Macroscopic)
• Aspergillus flavus macroscopically appears as yellow green velvety
colonies with reverse white on SDA agar- rapid grower
4) Aspergillus flavus (Microscopic)
• Lactophenol cotton blue mount showing thin hyaline septate hyphae
conidophore with globular vesicle.
• Conidia arise from the entire vesicle and are hyaline

5) Aspergillus fumigates (Macroscopic)

• Sabourad’s dextrose agar slope with smoky green velvety powder with
reverse white- rapid grower
6) Aspergillus fumigates (Microscopic)
• Lactophenol cotton blue mount showing
- Thin hyaline septate hyphae
- Conidiophores arise from hyphae at acute angle which end at conical
shaped vesicle
- Single row ( uniseriate ) phialides
- Conidia are hyaline and occupies upper third of vesicle

7) Cryptococcus-Negative staining
• Cryptococcus negative staining to demonstrated capsule
• Capsule appears as refractile delineated clear space surrounding the round
budding yeast cells against a black background

8) Candida- Gram stain

1. Gram positive oval budding yeast cells with pseudo hyphae seen.
2. Shape of conidia varies with species
3. Size- 3-6 um to 6-10 um

VACCINES

1) Hepatitis B vaccine
• Type – recombinant subunit vaccine
• Route of administration – Intramuscular over deltoid region (in infant
anterolateral aspect of thigh)
• Schedule
• For adults – 3 doses given at 0,1,6 months.
• Under national immunization schedule – zero dose at birth,6, 10 and 14
weeks along with DPT & Hib vaccines.

2) Mr Vaccine
• Type – live attenuated bivalent vaccine
• Strains used – Schwartz (measles), RA 27/3 (Rubella)
• Dose / route of administration – 0.5ml/subcutaneous
• Schedule
• 2 doses at 9 months & 12-15 months of age.

3) RABIES VACCINE
• TYPE – Purified Vero cell vaccine (cell – culture derived vaccine)
• Route & schedule – intra dermal (Thai red cross schedule) [ 2-2-2-0-2] for
post exposure prophylaxis
• 0.1 ml reconstituted vaccine is injected on two sides per visit on days 0,3,7,
and 28.

4) Pentavalent vaccine
• Type – combined vaccine contains diphtheria toxoid, pertussis vaccine
(whole cell/ acellular), tetanus toxoid, hepatitis b subunit and Hib
(Haemophillus influenzae B) vaccine.
• Route of administration: intramuscular – anterolateral aspects of thigh
• Schedule – 3 doses at 6,10,14 weeks of age under national immunization
programme.

5) BCG vaccine
• Bacillus Calmette Guerin Vaccine is a live attenuated vaccine for
Mycobacterium tuberculosis given at birth
• Danish 1331 strain is used in India
• It is reconstituted with normal saline and administered intradermally just
below the insertion of deltoid on left side.
• After vaccination a papule develops at 2-3 weeks, then at 5 -6 weeks
shallow ulcer is formed and finally scar is formed at 8 -14 weeks.

6) OPV VACCINE
• Oral polio vaccine is a live attenuated oral vaccine previously given under
UIP at birth,6,10,14 weeks and booster at 16-24 months
• OPV can be washed away if given immediately before or after breast
feeding
• Provides gut and herd immunity
• OPV can cause paralytic polio and vaccine derived polio viruses
• Gives long-lasting immunity
• Economical and safe.

7) Td vaccine
• Tetanus toxoid + Diphtheria toxoid (2 Lf)
• It is a toxoid vaccine given in a dose of 0.5 ml deep intramuscular injection
at 10 and 16 years.
• Prevents Clostridium tetani infection – tetanus
• Additional doses of tetanus toxoid are usually required in cases of open
injuries

8) Rotavac (rota virus vaccine)

• Live attenuated rotavirus 116E (G9P[11])
• Manufactured by Bharat biotech India
• 3 doses at 6,10,14 weeks along with pentavalent
• No vaccine induced intussusception reported

9) DPT vaccine
• Combined vaccine with Diphtheria, Pertussis (can be whole cell / acellular)
and Tetanus toxoid.
• Pertussis component acts as adjuvant to increase the immunogenicity of TT
and Diphtheria
• DPT booster given atb16-24 months and 5-6 years
• 0.5ml intramuscular route

10) TYPHOID VACCINE

• Typbar TCV® is a vaccine containing polysaccharide of Salmonella typhi
Ty2 conjugated to Tetanus toxoid.Vi capsular polysaccharide of Salmonella
typhi.
• 0.5 ml given intramuscularly to infants more than 6 months of age single
dose. Booster dose may be given after 3 years.
 11) Japanese encephalitis vaccine
• Live attenuated vaccine SA 14-14-2
• Under UIP given at 2 doses – 9 months completed and 16-24 months
• 0.5ml /dose subcutaneous at left upper arm