SPOTTERS
INSTRUMENTS
1. Seitz Filter
 Filtration is one of the physical method of sterilization used for heat labile substances
 It is used to remove the bacteria from heat labile liquids such as serum, sugar solutions and
antibiotics.
 This is a type of asbestos filter with high adsorbing capacity
 It contains single use, disposable discs which can be changed after each use
2. Mc Intosh Filter Jar
 It is an anaerobic jar, widely used for the cultivation of anaerobic organisms
 It contains of a stout metallic jar with a lid
 Lid consists of gas inlet and outlet, two electrical terminals
 The catalyst used is palladinised asbestos, which is hanged from the lid
 At one side it contains a tube with Methylene blue which acts as an indicator
 The streaked plates are kept inside using a stand and closed
 This is the best methods used to grow anaerobic organisms
3. Throat Swab
 Swabs are used to collect the clinical specimens
 These are made up of sterile cotton
 Swabs are also used for antibiotic sensitivity testing
 After preparing the swab using a stick, these are placed in a test tube and sterilized by using hot
air oven at 160 C for one hour
 E. g. Throat swab, wound swab etc
4. West’s’ Post nasal swab
 It is a cotton swab on a bent wire.
 The prepared swab is kept in a test tube and sterilized by using hot air oven
 It is mainly used in the collection of secretions from posterior pharyngeal wall
 It is mainly used in the case of whooping cough
5. Nichrome wire loop
 Wire loop is used in inoculation of clinical specimens onto the culture media
 It contains a handle to hold and nichrome wire loop to culture the specimen
 Sterilization is by Flaming using Bunsen burner
 It is also used in the hanging drop preparation and transfer of organism
STERILE MEDIA
6. Agar Agar
 It is prepared from a sea weed (Algae Geladium species)
 It contains long chain polysachharide and small amounts of protein like material and inorganism
salts
 Available as long shreds or powder form
 Used in concentration of 2 -3 %
 It melts at 98 C and solidified at 42 C
 It does not provide any nutrition to bacteria but acts as solidifying agent
7. Peptone water
 It is a basal liquid media
 All the non fastidious bacteria can grow in this media
 It contains peptone, NaCl and distilled water
 It is mainly used to
o See motility of the bacteria
o To test for the production of indole
o To check antibiotic susceptibility testing
o To prepare sugar media
8. Alkaline peptone water
 It is a liquid media similar to peptone water but the pH of the medium is adjusted to 8.4 – 8.6
 Vibrio cholerae resist alkaline pH and grows
 This medium acts as a selective and enriched medium for vibrio cholerae
 After inoculation subculture should be made after 6 hours onto solid media to differentiate the
organism
9. Robertson’s cooked meat medium
 It is a liquid anaerobic medium used for the cultivation of anaerobic organisms
 It is composed of meat particles and glucose broth
 Unsaturated fatty acids present in the medium absorb the oxygen within the bottle and creates
anaerobic condition
 Saccharolytic organisms turn the meat particles into pink colour and proteolytic organisms turn
the meat particles black
10. Nutrient agar
 It is a simple solid media used for the cultivation of non fastidious organism
 It consists of peptone, beef extract, NaCl and distilled water
 2% agar is added to solidify the medium
 It is used as a basal medium
 It is used to see the pigmentation and to perform antibiotic sensitivity testing
11. Blood agar
 It is an enriched media
 It is prepared by the addition of 5 – 10% sheep or horse blood to nutrient agar
 It is used to see the hemolysis or hemodigestion of RBC present in the media by the bacteria
itself or by the production of hemolysins
 Clear zone surrounding the colony indicates hemolysis
12. Mc Conkey’s agar
 It is a selective and differential media
 It contains peptone, NaCl, Sodium tauracholate, lactose, agar and distilled water
 The indicator used is neutral red
 It allows only enterobacteriaceae family members to grow which resist bile salts
 It differentiate lactose fermentors from non lactose fermentors
 Lactose fermentors appear pink in colour and non lactose fermentors appear without any colour
13. TCBS agar
 Thiosulphate citrate Bile salt sucrose agar
 It consists of yeast extract, peptone, Na2S2O3, NaCl, sodium citrate, sucrose etc.
 It is a selective media for Vibrio cholera
 Thiosulphate and bile salts inhibits the growth of all the organisms except vibrio
 Vibrio by fermentation of sucrose forms yellow colour colonies
14. Wilson & Blair Medium
 IT is a selective media used for isolation of typhoid and paratyphoid organisms
 It consists of bismuth ammonia citrate, sodium sulphite, sodium phosphate, brilliant green etc
 Brilliant green inhibits E. coli and other organisms
 By reducing bismuth and sodium sulphite to silphide salmonella forms black colonies
 It is mainly used for isolation of salmonella from feces.
15. Chocolate Agar
 It is prepared by adding blood to nutrient agar, Aix well and heat at 90 C for 10 mins and pour
into the plates
 By heating haemoglobin is liberated from the disrupted cell. The globin part will be destroyed by
heat and the haem portion will diffuse into the media
 This media is mainly used for those organisms which require ‘X’ factor, which is present in haem
 It is mainly used to grow Haemophilus, Pneumococci etc
16. Lowenstein Jensen’s Medium
 It is the media used for the cultivation of Mycobacterium tuberculosis
 It consist fo egg yolk, mineral salt solution and malachite green and glycerol
 Sterilization of the medium is by inspissation
 It is a solid medium with our agar. Egg yolk solidifies the medium
 Malachite green inhibits the other bacteria
 Colonies of Mycobacterium are dry, rough and creamy in colour.
17. Castaneda’s Biphasic Medium
 Brain heart infusion broth and agar media
 It is a biphasic media used to grow salmonella
 It is an alternative to glucose broth and bile broth to avoid contamination by frequent sub
cultures.
 Broth part indicates the growth by turbidity and solid part by colony formation
18. Sabourauds Dextrose agar
 SDA is used for primary isolation of fungi
 Its pH is 5.0
 Sometimes Chloramphenicol is added to suppress the growth of contaminated bacteria and
Cycloheximide is added to suppress the contaminating fungi
19. Glucose broth
 It is a liquid media used to grow pyogenic bacteria
 It consist of nutrient broth with 0.5% sterile glucose
 Growth can be seen by turbidity or by granular appearence
20. Bile broth
 It is a liquid media used to grow salmonella
 It is mainly used for blood culture
 It consist of nutrient broth and 0.5% sodium tauracholate
 Sodium tauracholate inhibits most of the organisms and allow salmonella to grow
 After overnight incubation sub culture on to other solid media to see the colony morphology
 Growth is indicated by turbidity of the medium
21. Loeffler’s Serum Slope
 It is composed of glucose broth with horse/ sheep . ox serum
 It is a enriched medium useful in the cultivation of Corynebacterium diphtheria
 It is a solid medium and serum solidifies the medium on heating
 Colonies of Corynebacterium spears within 5 – 6 hours
 Sterilization of the medium is by inspissation
22. Selenite F Medium
 It is an enrichment medium
 It enriches the growth of salmonella and Shigella species
 It inhibits the growth of other colofirm bacteria
 Salmonella paratyphi A and some Shigella species fail to multiply in this medium
 The growth of the organisms will be indicated by the turbidity
23. Tetrathionate broth
 It is an enrichment medium
 It enriches the growth of Salmonella and Shigella species.
 It allows proteus species to grow
 The medium contain the indicator Phenol Red
 It is dispensed in screw capped bottles
MEDIA WITH GROWTH
24. Nutrient agar with Staphylococci
 Different strains of staphylococci produce different pigments on nutrient agar
 Staphylococcus aureus produces Golden yellow pigment
 Staphylococcus albus produces white pigment
 Staphylococcus citreus produces Lemon yellow pigment
 The pigment of staphylococci is believed to be a lipoprotein allied to carotene
 Pigmentation of bacteria is seen mostly on nutrient agar
25. Blood agar with Proteus
 Proteus show spreading type of growth on Blood agar
 This is called swarming
 The swarming between identical strains is overlapped
 And the swarming between non identical strains were inhibited and this is called Diene’s
phenomenon
26. Wilson Blair’s agar with Black colonies
 Black colonies on Wilson Blair agar are formed by Typhoid and Paratyphoid organisms
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Brilliant green present in the medium inhibits the growth of other organism
Typhoid and paratyphoid organisms reduce sodium sulphite to sulphide along with bismuth
sulphite to bismuth sulphide
 Organisms in presence of bismuth sulphide and glucose forms black colonies
 Salmonella typhi forms black colonies within 24 hours
 Salmonella Paratyphi B forms black colonies after 24 hours
27. Tellurite blood agar with black colonies
 Black colonies on tellurite blood agar are formed by Corynebacterium diphtheria
 Tellurite, which is present in the medium inhibits the growth of other bacteria
 Corynebacterium diphtheria reduce tellurite to metallic tellurium which is incorporated in the
colonies, giving them grey or black colour.
 Different modifications of tellurite blood agar are used. Examples ---- Mc Leod’s and Hoyle’s
Medium
28. Mac Conkey agar with lactose fermenting colonies
 Bacteria which grown on Mc Conkey’s are classified into Lactose fermentors and non lactose
fermentors.
 Lactose fermentors are pink in colour
 Lactose fermenting bacteria utilize lactise and produce acids.
 The indicator neutral red gives red / pink colour in acidic pH.
29. Mac Conkey’s agar with Non lactose fermenting colonies
 Bacteria which grow on Mac conkey’s agar are classified into Lactose fermentors and Non
lactose fermentors
 Non lactose fermentors appear colourless
 Non lactose fermentors cannot utilize lactose in the media and no acids are formed.
 Hence there is no change in the pH of the medium and also no effect on the neutral red making
colonies appear colourless.
30. Lowenstein Jensen Medium with growth
 It is mainly used to grow human tubercle bacilli.
 Tubercle bacilli grow as heaped up, dry, yellow colonies.
 Tubercle bacilli obtain their nitrogen from the asparagine and carbon from glycerol
 Malachite green helps to inhibit the growth of other organism
31. Sabouraud’s Dextrose agar with Candida
 SDA is basal media to grow fungi
 Candida on SDA grows as like bacteria on nutrient agar
 Colonies are creamy white, Moist, non pigmented.
 The inoculated tubes and plates will be incubated at 25 C
32. Sabouraud’s Dextrose agar with Aspergillus niger
 Colonies at first appear as cottony white or faint yellow medium
 On further incubation colonies appear as black. Crowded conidial structures.
 Conidial structures soon cover all the margins of the margins of the developing colonies
 No pigment is produced on the reverse side of the media
33. Nutrient agar with Pseudomonas
 Pseudomonas on Nutrient agar produce a number of pigments
 Common pigments seen are pyocyanin which appears bluish – green ion colour and produced by
Pseudomonas aeruginosa only
 Fluorescein which appears greenish yellow in colour and produced by many other species also.
 In old cultures these pigment may be oxidized to yellowish brown pigments.
34. Antibiotic sensitivity testing plate
 Antibiotic sensitivity testing is done to check the in vitro activity of the drug
 It is done on Mueller Hinton agar medium
 The method used is Kirby Bauer method
 The sensitivity or resistance of the organism will be seen by measuring the zone of inhibition in
millimeters.
 Depending on the concentration of the drug the zone varies
 The result will be taken as sensitive, resistant or intermediate sensitive.
BIOCHEMICAL REACTIONS
35. Indole test
 This test is to determine the ability of the organism to decompose amino acid tryptophan into
indole
 The organism is inoculated in peptone water and incubated at 37 C overnight
 Next day add Kovac’s Reagent (Para dimethly animo benzaldehyde) 0.5ml to the broth
 POSITIVE TEST: Red colour ring at the surface of the medium e.g. E. coli, Proteus vulgaris
 NEGATIVE TEST: yellow colour ring at the surface of the medium e.g. Klebsiella . Proteus
mirabilis
36. MR Test
 To detect the production of acid during the fermentation of glucose and maintenance of pH below
4 .5
 The organism is inoculated into glucose phosphate broth and incubated at 37 C for 2 – 5 days
 Add 0.04% solution of MR reagent 5 drops into the broth culture and mix well.
 POSITIEV TEST: Red colour e.g E. coli, Yersinia
 NEGTAIVE TEST: yellow colour e.g. Klebsiella and enterobacter
37. VP Test
 To detect the production of acetyl methyl carbinol from pyruvic acid as an intermediate stage in
its conversion to 2 -3 butylene glycol.
 The glucose phosphate broth is inoculated with the test organism and incubated for 48 hours
 Add 0.6ml of 5% solution of alpha Naphthol in ethanol and add 0.2 ml of 40 % KOH to I ml of
glucose phosphate medium culture
 POSITIVE TEST: Pink colour within 2 – 5 mins deepening to crimpson colour in 30 mins. E.g.
Klebsiella and Enterobacter
 NEGATTIVE TEST: colourless for 30 mins e.g. E. coli and Micrococcus
38. Citrate test
 To determine the ability of an organism to utilize citrate as sole sources of carbon resulting in
alkalinity
 Both solid and liquid media can be used
 Solid is Simmon’s citrate and Liquid is Koser’s medium
 Bacterial colony is streaked on to the Simmon’s citrate agar
 Incubate at 37 C overnight
 POSITIV TEST: It turns blue from green e.g. Klebsiella and Pseudomonas
 NEGATIVE TEST: No change ion colour e.g E. coli
39. Urease Test
 It is used to see the urease positive and urease negative reaction of the bacteria.
 The positive reaction depends on the production of an enzyme UREASE.
 Urease decomposes urea resulting in the formation of ammonia which is detected by colour
change of the media.
 The media used is christensen’s urease agar with phenol red as the indicator.
 POSITIVE TEST: Pink colour media. E. g. Proteus, Klebsiella
 NEGATIE TEST: No colour change (Yellow colour) e.g. E. coli, Pseudomonas
40. Coagulase Test
 It is performed to detect whether a bacteria produce Coagulase enzyme or not.
 It is detected by using slide and tube Coagulase test
 Slide Coagulase test detects bound Coagulase and tube Coagulase test detects free Coagulase.
 In this dilited human or rabbit plasma is used to which the organism suspension is added and
incubated for 3 – 6 hours.
 Clotting occurs in positive cases
 Clotting is absent in negative cases
 This is the test used to differentiate Staphylococcus aureus from other Staphylococci.
41. Sugar reactions of E. coli
 Sugar media help in identification of bacteria
 Sugar media contains 1% sugar in peptone water along with the indicator (Andrade’s indicator)
 The tube consist of a inverted durham’s tube which will be used to detect the gas production
 The Escherichia coli ferments glucose, lactose, maltose and Mannitol except sucrose
42. Sugar Reactions of Klebsiella
 Sugar media help in identification of bacteria
 Sugar media contains 1% sugar in peptone water along with the indicator (Andrade’s indicator)
 The tube consist of a inverted durham’s tube which will be used to detect the gas production
 The Klebsiella species ferments glucose, lactose, sucrose, maltose and Mannitol .
PARASITOLOGY
43. Ancylostoma duodenale – male
 It is called Old world hook worm
 It causes hookworm disease or Ancylostomiasis
 Adult worm reside in small intestine and duodenum
 These are small greyish white cylindrical forms appear reddish brown in colour
 Anterior end is lsightly bent hence it is called Hook worm
 Infection if by filariform larvae
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No intermediate host present
Sexes are separate
MALE:
o Smaller than female worm and 8 mm in length
o Posterior end is expanded in an umbrella like fashion which is called copulatory bursa
o Genital opening is present posterior with the cloaca
44. Ancylostoma duodenale – female
 It is called Old world hook worm
 It causes hookworm disease or Ancylostomiasis
 Adult worm reside in small intestine and duodenum
 These are small greyish white cylindrical forms appear reddish brown in colour
 Anterior end is lsightly bent hence it is called Hook worm
 Infection if by filariform larvae
 No intermediate host present
 Sexes are separate
 FEMALE:
o It is longer than male and is about 12.5mm in length
o Posterior end in tapering without copulatory bursa
o Genital opening is at the junction of posterior and middle third of the body.
o After fertilization female worm lays eggs.45. Ascaris lumbricoides – Male
45. Ascaris lumbricoides – Male
 It is also known as round worm
 It is the parasite of lumen of the small intestine
 It is the causative agent of ascariasis
 Infection is by ingestion of contaminated food with eggs
 Adult worms are rounded and tapers at both ends
 MALE:
o It measures about 15 – 25 cm in length and 3 – 4 mm in breadth
o Posterior end is curved. Genital pore opens into the cloacae with two curved copulatory
spicules
46. Ascaris lumbricoides – Female
 It is also known as round worm
 It is the parasite of lumen of the small intestine
 It is the causative agent of ascariasis
 Infection is by ingestion of ocnatmnated food with eggs
 Adult worms are rounded and tapers at both ends
 FEMALE:
o It is longer and stouter than male
o It measures about 25 – 40 cms in length and 5mm in breadth
o Posterior end is straighter and pointed
o Anus opens on the ventralaspect as the transverse slit
o Vulva opens at the junction of the anterior and the middle thirds of the body
o Fertilized female worm lays eggs
47. Tapeworm
48. Enterobius vermicularis
 Also known as pinworm or seat worm or thread worm
 Worms can be recovered by collecting eggs from perianal skin or NIH swab
 Worms are small measuring 8 – 13mm in length, white in colour resembling a small piece of
thread.
SLIDES
49. Staphylococci
 It is gram positive cocci arranged in clusters
 The cocci are arranged in clusters due to division of parent cell in all the planes
 The size of staphylococci is 1 x 0.5µm approximately
50. Streptococci
 Streptococci are gram positive cocci arranged in chains
 The arrangement is due to division of the cocci in transverse plane and the daughter cells unable
to separate from the parent cell, hence they are arranged in chains
 They can be Streptococcus. pyogenes or Str. viridans
51. Mycobacterium tuberculosis – ZN stain
 The long slender pink colour bacilli are Mycobacterium tuberculosis
 The slide is stained by ZN method as the back ground and all the other cells are blue in colour
 Sometimes beaded appearance can be observed
 Barred or broken forms of Mycobacterium tuberculosis is observed when the patient is under
treatment
 The grading of the smear is 3+
52. Corynebacterium diphtheria – Albert’s stain
 The bacteria are arranged in X.L.M.V arrangement
 The polar or metachromatic bodies are stained bluish purple
 The bacillary body takes up the malachite green and appears green in colour.
53. Candida – Grams stain
 The oval gram positive budding cells are Candida
 It is a yeast like fungi
 The presence of pseudohyphae in direct smear indicates the invasiveness of the yeast.
54. Cryptococcus neoformans – Grams stain
 It is a spherical gram positive budding yeast cell
 Covered with a clear halo indicates capsule
 It is a true yeast
 It causes cyrptococcal meningitis in immunocompromised patients
55. Microfilaria
 It is a delicately coiled larval form of Wuchereria bancrofti
 It is sheathed larvae with sheath loosely arranged and extends beyond the body
 The nuclei are arranged from head to tail tip
 A nerve ring is arranged at the junction of anterior 1/3 to posterior 2/3 rd
56. Plasmodium falciparum gametocyte
 The gametocytes of Plasmodium falciparum are crescent or sickle shaped
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MICROGAMETOCYTE:
o The microgametocyte is broader, shorter and have blunt ends
o The cytoplasm stains light blue
o The nucleus is scattered in fine granules over a wide area
 MACROGAMETOCYTE
o It is longer, narrow and have pointed ends
o The cytoplasm stains deep blue
o The nucleus is condensed into a small compact mass at the center
57. Rhizopus – LPCB Mount
 It belongs to zygomycetes
 The hyphae are aseptate
 Sporangium is borne on the sporangiophore
 The sac is called sporangium and contains sporangiospores
58. Aspergillus niger – LPCB Mount
 The hyphae are septate and are dichotomously branched
 Contains conidia borne on conidiophores and arranged like balls
 The conidia are black in colour hence the name niger
SEROLOGY / IMMUNOLOLGY
59. Widal Test
 It is used to detect the presence of antibodies against Salmonella typhi and paratyphi “O” and “H”
antigens.
 It is quantitative test and uised to determine the titer of antibodies
 In this test 4 rows of tubes arranged and the serum sample is serially diluted
 To the first row of the tube “TO” antigen is added and to the second tow “TH” antigen is added.
 To the 3rd row AH and to the 4th row BH antigens are added.
 For each antigen one control is arranged without a sample.
 After the procedure tubes are incubated at 37 C and for 24 hours.
 POSITIVE TEST for O antigen: Dic like powdery granular deposit
 NEGATIVE TEST for H antigen: Loose cotton wooly fluffy agglutination.
60. VDRL Test
 It is considered as standard test for syphilis
 It is a slide flocculation test. In this the antigen used is cardiolipin.
 In this test the serum sample is inactivated by heating at 56 C for 30 minutes.
 On a VDRL slide the inactivated serum is mixed with cardiolipin antigen and rotated for four
minutes.
 Positive reaction is indicated by clumping (floccules) formation.
 Negative reaction is indicated by the absence
61. ELISA Test
 It is an antigen – antibody reaction, used to determine the quantity of antigen or antibody from the
given serum sample.
 It is used in the identification of infectious agent.
 It is time taking procedure and contains…
o Sample diluents: to dilute the serum sample.
o Conjugate: to stabilize the bond between the antigen and antibody
o Substrate: to give colour reaction to the antigen.
o Stop solution: To stop the reaction between the Ag and Ab
 Result can be taken by using microassay plate reader.
 Sample with the titre above than the cut- off value is considered as positive and below than that as
negative
VIROLOGY
62. Embryonated Egg
 Embryonated egg is used for the cultivation of viruses
 Several sites of embryonated egg are used in the cultivation
 Chorioallantoic membrane inoculation produces visible reactions
 Allantoic membrane is inoculated for the preparation of vaccines
 Amniotic sac inoculation is employed for the primary isolation of influenza viruses
 Yolk sac inoculation is used for the cultivation of Chlamydiae and ricketssiae
Common questions asked for the Gram staining
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Theories of gram staining
Reasons for gram variability
Cell wall deficient forms
What are “L” forms , how did they get their name .
Difference between L forms and Mycoplasma
Difference between Protoplast and spheroplast
Comment of gram staining on growth curve
E.gs of gram positive cocci, gram negative cocci
E.g of Gram positive bacilli and gram negative bacilli
Stains used in grams staining procedure
o Methly violet , other stains
o Mordent definition, e.g.
o Decolourizer – Acetone, alcohol and their timings
o Counter stain – other stains
Common questions asked for special stains
ZN Stain
 Procedure of ZN staining , principle
 Cell wall structure if Mycobacterium tuberculosis
 Cold staining method ( Kinyon’s staining)
 Modified acid fast staining method , organisms using different conc. of decolorizing with
sulphuric acid
 Morphological difference between mycobacterium tuberculosis and Mycobacterium bovis
 Concentration techniques
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Grading of the smear
Presence of min number of the tubercle bacilli in the sputum to be positive by ZN staining
technique
Rapid staining techniques to screen many samples in the lab – Fluoroscent staining method with
Auromine O
How many min no of bacilli to be present in sputum to be positive by culture method
Different culture media to grow the Mycobacterium tuberculosis
Generation time for M. tb , time taken to get a positive culture in LJ media
Composition of LJ media and how do you sterilize it
Rapid culture techniques of M. tb
Antimybacterial susceptibility testing.
Albert’s stain
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Composition of Albert’s I stain
Composition of Albert’s II stain
Method of staining
Other special stains to stain metachromatic bodies
Describe the arrangement of C. diphtheria and reasons for its arrangement
Other names for the metachromatic granules
After sending throat swab for Albert’s stain do you want to wait for treatment?.
Can you confirm diphtheria by a positive Albert’s stain alone?
Reason for the toxin production in Corynebacterium diphtheria
Culture methods for Corynebacterium diphtheria
LSS – after how much time growth is observed
Tellurite blood agar – After how much time growth is observed
Basis of classification of C. diph
Invivo and invitro toxigenicity tests