SPOTTERS LIST DISCUSSION

MLT 2 ND YEAR
INSTRUMENTS(10)
 1.SINTERED GLASS FILTER

• Made of fused powdered glass

• Uses- to filter heat labile fluids- Sugar
solutions, antibiotic solutions,
• Disadvantage- fragile
• Method of cleaning-reverse flow of water
 2.CANDLE FILTER

• Physical method of sterilization- Filtration

• Bacterial filter
• Types- 1.Unglazed ceramic filter- Chamberland & Doulton
filters
• 2. Diatomaceous earth filter- Berkefeld & Mandler filters
• Advantage- Simple and easy to use
• Disadvantage- fragile
• Uses- purification of water for domestic and industrial
purpose
• Method of cleaning-Boil in water for 30mints and clean the
surface
 3.TUBERCULIN SYRINGE
• Used for Tuberculin or Mantoux test
• Used to deliver 0.1 ml of PPD- 5TU intradermally in the
flexor aspect of the forearm
• Readings taken 48-72 hrs later for induration
• POSITIVE ≥10mm
• NEGATIVE ≤5mm
• EQVIVOCAL 6mm -9mm
• Pre packed single use disposable
syringes sterilized by γ radiation
 4.MAC INTOSH FILDE’S JAR

• Anaerobic culture method

• Used to culture anaerobic bacteria like Clostridium spp,
Bacteroides spp, etc
• Made of stout glass or metal jar with lid which can be
clamped tight
• Lid has-
• Inlet- for hydrogen gas
• Outlet- To create vacuum
• Two terminals for electric connection, below with
porcelain spool for palladinised asbestos – catalyst
• Catalyst-
• a. Old method- Palladinised asbestos, alumina pellets coated
with palladium,
• b. New method-Gaspak- it’s a disposable envelope containing
chemicals which
• generate hydrogen & carbon dioxide gas which in presence
of cold
• Catalyst produces anaerobic environment
• Advantage of Gaspak- simple,effective & eliminates creation
of vacuum
• and use of hydrogen gas
• Indicator- Methylene blue or Reazurin
 5.WIDAL RACK WITH TUBES

• WIDAL TEST- diagnosis of enteric fever

• Dryers tube- narrow tube with conical bottom for
H agglutination- loose cottony clumps
• Felix tube- round bottom for O agglutination-
chalk powder like deposit at the bottom of tube
• Significant titer for O agglutination 1:100 and
above
• Significant titer for H agglutination 1:200 and
above
6.MICROTITRE PLATE

• A flat plate with 96 wells used as small test

tubes which are precoated with antigens,
antibodies or proteins
• Used in enzyme-linked immunosorbent assay (
ELISA), TPHA, and H I A.
• Composition- Polystyrene, Polypropylene,
Polycarbonate, Cyclo-olefin.
• The earliest microplate was created in 1951 by
a Hungarian, Dr. Gyula Takátsy
7.INOCULATION LOOP

• Types
• Pre packed γ radiation sterilized single use disposable plastic
loops
• Reusable metal loops
• Metal loops made of- platinum or nichrome or tungsten or
stainless steel. They are sterilized by red hot method
• Thickness- 26-27 gauge (standard wire gauge-SWG)
• Length-4-6 cm
• Diameter of loop-2-4mm
• Loop holder made of steel
• Use to inoculate specimens on culture media
 8.PASTURE PIPETTE

• Used to transfer small quantities of liquids

media or reagents.
• Types
• Glass tubes tapered to a narrow point, and
fitted with a rubber bulb at the top.
• Plastics are single use disposable pipettes
9.STERILE SWAB

• Cotton tipped with plastic or wooden handle in a plastic

tube
• Pre packed cotton swabs are sterilized by γ radiation (Cold
sterilization)
• Uses- To collect throat swab, ear swab, nasal swab, wound
swab
• Modifications-
• Serum tipped sterile swab for delicate organisms like
Streptococci,
• C. diphtheria
• Calcium alginate swabs- For Neisseria spp.
 10.SPECIMEN COLLECTION CONTAINER

• Pre sterile plastic disposable container with

screw cap
• Sterilized by γ Radiation
• Uses- To collect clinical specimens eg- Sputum,
Stool, Pus etc
CULTURE MEDIA-
UNINOCULATED
CULTURE MEDIA-
INOCULATED
 NUTRIENT AGAR

• Simple Media. uninoculated

• Composition.
• Peptone, meat extract, yeast extract, sodium chloride, agar (2-
3%).
• USES-
• 1. Demonstrate colony morphology.
• 2. Demonstrate pigment production- S. aureus- golden yellow
non- diffusable
• pigment, Pseudomonas aeruginosa- blue-green diffusible
pigment.
• Nutrient Broth- Same as nutrient agar media without agar.
• Semisolid Nutrient agar-Same as nutrient agar with agar- 0.5-1%.
 BLOOD AGAR

• Enriched media uninoculated

• Composition -Nutrient agar with 5-10% sheep blood
• Indicator media-
• α hemolysis- Green zone around colony- S. viridans
• β hemolysis - Clear zone around colony- S. aureus
• γ hemolysis- No zone around colony- Enterococci
• Used to cultivate fastidious bacteria like Streptococcus
 CHOCOLATE AGAR
• Enriched media uninoculated

• Nutrient agar is heated up to 70- 820 C, and 5-

10% sheep blood is added, the color turns
from red to brown (chocolate color).
• Used to cultivate fastidious bacteria like H.
influenza, C. diphtheria, Neisseria and
Streptococcus species.
 MAC CONKEY AGAR

• Differential & Indicator media

• Selective media- allows growth of Enterobacteriaceae
• Composition- Peptone, Lactose( Sugar), Agar- Solidifying agent,
Neutral red- Indicator, Taurocholate- Selective agent
• Principle- Lactose fermentation leads to acid production & neutral red
changes to pink color.
• COLONY TYPES- LF- Lactose fermenters
• NLF- Non Lactose fermenters
• LLF- Late Lactose fermenters

LF-Pink, E. coli,Klebsiella spp

NLF-Colourless, Shigella spp,Salmonella spp, Proteus spp,Pseudomonas
LLF-Colourless to pink, V. cholerae
 LF NLF

MAC CONKEY WITH LF/NLF

MAC CONKEY-uninoculated
 TCBS

• TCBS- Thiosulfate Citrate Bile salt Sucrose agar

•
• COMPOSITION-
•
• Sodium thiosulfate , Sodium citrate, Selective agent-Bile salt, Sugar- Sucrose,
Indicator- Bromothymol blue
•
• PRINCIPLE-
•
• Media inhibits the growth of coliforms allowing Vibrio species to grow.
• V. chloerae ferments sucrose which changes the indicator to yellow colour, hence
imparting a yellow colour to the colonies. Other vibrios e.g., V. parahemolyticus
remain green (Non sucrose fermentor)
•
• Selective media for V. cholera (Plating media)
TCBS –Unicoluated TCBS -Incoluated
 L J MEDIUM

• Selective medium for M. tuberculosis

• Culture more sensitive than microscopy, can detect 10-100 viable organisms/ml of
sputum
• Composition- coagulated hen’s egg, asparagine, mineral salt solution, malachite green,
glycerol, sodium pyruvate, pH- 6.9
• Malachite green- selective agent & gives colour to the medium
• Contains no agar, solidifying agent is egg
• Solidified by inspissiation
• Media stored in Screw cap bottles, in the form of slants
• Incubation period: 6-8 weeks (M. tuberculosis)
One week (atypical Mycobacteria)

• Colony Morphology:
• M. tuberculosis -Dry, rough, raised, irregular colony, with wrinkled surface, not easily
emulsified and white buff coloured (ROUGH, TOUGH, BUFF)
• M. bovis – Flat, smooth, moist, white colonies, breaks easily on touch
LJ –Unicoluated
LJ -Inoculated
 RCM-unicoculated

• Anaerobic liquid media

• Nutrient broth with predigested fat free cooked meat of ox heart
• 2-2.5 cm of meat particles, over lay with broth and seal with a layer
of sterile liquid paraffin.
• Principle for anaerobiosis.
• Unsaturated fatty acids of meat utilizes oxygen for auto oxidation
• Reducing agents in meat- Glutathione, cystine (Sulphydryl
compounds)
• Interpretation- Saccharolytic- meat turns pink -eg. C. perfringes
• Proteolytic - meat turns black- eg. C. tetani
 BLOOD CULTURE BROTH

• Brain Heart Infusion Broth

• Enriched media
• Used for isolation of bacteria or fungus from blood
• Quantity-
• Adult- 50-100 ml of broth: 5-10 ml of blood
• Pediatric-25 ml -30 ml: 2.5- 3 ml blood
• Blood: Broth-1: 10
• Dilution is done to-
• Counteract the bactericidal action of blood- Like complement and
antibodies
• To dilute the antibiotics in the blood
• Incubation period- overnight
• Primary and Secondary subculture done consecutively
 BACTEC-BLOOD CULTURE BOTTLE

• BACTEC bottles are used to detect the presence of aerobes,

anaerobes, fungi, yeast, and mycobacteria in blood samples using
automated system.
• Yellow colored capped bottles are used for paediatrics.
• Green colored capped bottles are used for adults.
MILK AGAR- S AUREUS , S ALBUS, S. CITREUS

• S. aureus ,S. albus,S.citrus

• Culture media- Nutrient agar showing Staphyloccal
pigments
• Colony morphology- White- yellow opaque circular
colonies, 2-4mm diameter, regular margin, easily
emulsifiable
• Types of pigments-
• S. aureus- golden yellow pigment
• S. albus- no pigment- white coloured colonies
• Pigment- Nondiffusable, lipoprotein allied to carotene
• Pigment production enhanced at 22 0 C, aerobic
environment, 15 glycerol monoacetate or milk( milk agar)
S. citreus S. albus
 POTASSIUM TELLURITE- C DIPHTHERIA

• Selective media for the isolation of C. diphtheriae

•
• Composition- Chocolate agar with 0.04% Potassium tellurite
•
• Principle:
• Potassium tellurite- prevents the growth of other bacteria but allows
• C. diphtheriae to grow which reduces tellutite to metallic tellurium
imparting a grey black colour to the colony.
• Incubation period- 48hrs
•
• Colony morphology- C. diphtheriae produces black metallic colonies
of 1-2mm diameter
Pottasium tellurite agar with growth
 WILSON BLAIR WITH GROWTH
• WILSON AND BLAIR / BISMUTH SULPHITE BILE SALT MEDIUM
• Selective and indicator media for Salmonella species
•
• Composition:
• Peptone, glucose, disodium phosphate, ferrous sulphate, bismuth sulphite indicator, brilliant green,
agar
•
• Principle:
• The bile salts in the medium inhibit the growth of coliforms in the feces; Salmonella reduces Bismuth
sulphite to bismuth sulphide, imparting a black metallic colour to the colonies
•
•
• Interpretation:
•
• Jet black colony with metallic sheen due to H2S production seen in all Salmonella spp. except S.
paratyphi A (Green colony)
•
• Salmonella typhi- Produces black colonies with black metallic sheen surrounding the colony after 18
hours.
• Salmonella paratyphi A -Produces green colour colonies
WILSON BLAIR WITH SALMONELLA TYPHI
 6.TCBS WITH GROWTH

• TCBS- Thiosulfate Citrate Bile salt Sucrose agar

•
• Selective media for V. cholera (Plating media)
•
• Composition:
• Sodium thiosulfate, Sodium citrate, Sugar- Sucrose, Selective agent-Bile salt
• And indicator- Bromothymol blue, p H- 8.6
•
• Principle:
• Media inhibits the growth of coliforms allowing Vibrio species to grow.
• V. chloerae ferments sucrose which changes the indicator to yellow and
imparting yellow colour to the colonies.
• Other vibrios e.g., V. parahemolyticus remain green (non sucrose fermentor)
 7.PROTEUS ON BLOOD AGAR

• Nutrient agar plate showing swarming growth.

• Swarming growth- is due to to actively motile bacteria which spreads on the
surface of agar plate in successive waves to form a thin filmy layer in concentric
circles
• Organisms which swarm- Proteus spp, Clostridium tetani
• Swarming is inhibited by-
• Agar 6%
• Alcohol 5-6 %
• Chloral hydrate- 1:500
• Sodium azide 1: 500
• Sulphonamides
• Boric acid 1:1000
• Surface active agents
• Mac Conkey agar inhibits swarming
 9.UREASE- POSITIVE & NEGATIVE

• Media is made as slants

• Principle- to demonstrate the ability of the organism
to produce urease enzyme, which splits urea into
ammonia and other alkaline compounds and changes
the colour of the indicator from yellow to pink colour.
• Composition- Urea, phenol red, agar, buffer salts, etc
• Positive urease test shows - Pink colour -Proteus spp,
Klebsiella spp
• Negative urease test shows - Light yellow colour-
Salmonella spp, E. coli
 10.CITRATE- POSITIVE & NEGATIVE

• Media is made as slants

•
• Principle- to demonstrate the ability of the organism to utilize
Citrate as the sole source of carbon
•
• Composition- Citrate, Bromothymol blue, agar, buffer salts, etc
•
• Positive citrate test- Blue colour- Klebsiella spp, all Salmonella
except
• Salmonella typhi
• Negative citrate test- Green colour -E. coli
• Other media used - KOSER’S CITRATE MEDIUM
11.TSI- A/A, K/A, K/A WITH H2S, UNINOCULATED TSI

• Type of media- Composite media with slant & butt

•
• Principle- To demonstrate the ability of the organism to utilize sugars like, glucose, lactose,
sucrose & produce gas & H 2 S
•
• Composition- Three sugars- Glucose: Lactose: Sucrose-10:1:1, Indicator- Phenol red, Ferric
salt- to indicate H 2 S production, Agar, Distilled water.
•
• Interpretation is done after 18-24 hrs as fallows
•
• Alkaline/ Acid (K/A)- Pink/ Yellow- only glucose is fermented- Shigella spp, Salmonella
paratyphi A
•
• Acid/ acid(A/A)- Yellow/Yellow- Lactose/ Sucrose ferments-
• E. coil (Little or no gas), Klebsiella (abundant gas seen)
• c) Alkaline/ Acid (K/A)- with black color at butt- Non lactose/ sucrose
 12.INDOLE TEST - POSITIVE & NEGATIVE

• Principle- demonstrates the ability of certain bacteria to

decompose the amino acid tryptophane to indole gas, which
accumulates in the medium.
• This indole is tested by colorimetric reaction with p- dimethyl-
aminobenzaldehyde
• Medium- peptone water
• Reagent- p- dimethyl-aminobenzaldehyde(Kovack’s reagent)
• Method- Inoculate medium, incubate 24-48hrs. Add 0.5ml of
Kovac’s reagent and shake gently.
• Interpretation-
• Indole positive- Pink ring at the top- Eg- E. coli
• Indole negative- No pink ring at the top-Eg- Klebsiella
SLIDES-BACT/PARA(11)
1.STAPHYLOCOCCI
2.STREPTOCOCCI
3.PNEUMOCOCCI- GRAM’S STAIN
4.GONOCOCCI
5.M. TUBERCULOSIS
6.M. LEPRAE
7.Corynebacterium DIPHTHERIAE- ALBERT’S STAINING