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Direct microscopy
- 2. HISTORY An early microscope was made in 1590 in Middelburg, Netherlands: Hans Lippershey and Hans Janssen. Giovanni Faber coined the name microscope
- 3. . Antoni van Leeuwenhoek who discovered red blood cells and spermatozoa and helped popularize microscopy as a technique. 9 Oct. 1676, Leeuwenhoek reported the discovery of micro-organisms. In 1893 August Köhler developed a key technique for sample illumination, Köhler illumination, which is central to modern light microscopy.
- 4. TYPE OF MICROSCOPE Light microscopes bright-field microscope dark-field microscope phase-contrast microscope fluorescence microscopes Others - transmission electron - scanning electron
- 5. ADVANTAGES Empirical choice of antibiotics can be made on the basis of gram stain’s report Choice of culture media for inoculation can be made empirically Evaluate the quality of specimen Co-relate the smear findings with that of culture
- 6. . Provide internal quality control when direct smear result are compared to culture result Accurate assesment of the WBC:RBC Visualise key morphological features Capsule- india ink Spiral shape of treponemes – dark ground microscopy
- 7. DIS-ADVANTAGE Less sensitive then culture method Doesn’t permit the identification of species Problems in stained preparations Carry over of organisms Thickness requiring varying decolouration times
- 8. TYPE OF MICROSCOPIC EXAMS WET PREPARATION • SALINE • IODINE • INDIAN INK • LACTOPHENOL BLUE STAINED PREPARATIONS • GRAM STAIN • Z-N STAIN • TRICHROME • CALCOFLOUR WHITE
- 9. URINE CAN BE DONE FOR:- Whether pus cells are present in number indicative of infection Screening out negatives-Rant- shepherd method When information urgently required
- 10. . Presence of casts, crystals rbc’s- non-infective condition Co-relate with the culture findings Terminal spined egg --Schistosoma haemtobium Microfilariae in chyluria- W. boncrofti
- 12. Pre processing Generally MSU any time For urethritis, prostatitis- initial flow of urine For AFB- EMU,3samples For wet film- 0.05ml + cover slip 1wbc/7hpf—significant pyuria
- 13. URINE WET MOUNT FAT DROPLETS CONTAMINATED
- 15. Interpretation of wet film Symptomatic + pus but N/G- Pus cells + squamous epithelial cells-
- 16. DRAWBACKS It is laborious and time consuming May yield misleading result if performed cursorily CONCLUSION- should be reserved for special investigation in selected cases.
- 17. SPUTUM PRE PROCESSING Homogenisation- sputum +dithiotheritol vortex for 15 sec stand for 15min.
- 18. . WET FILM:- For parasites For fungal elements GRAM STAIN:- Evaluate the quality of sputum:- WHO:- <10 PMN/epi. cells
- 19. FOR ADULTS :- >14 years TYPE OF CELLS COUNT GRADE NEUTROPHILS <10 0 10 - 25 +1 >25 +2 SQUAMOUS EPITHELIAL CELLS 10 - 25 - 1 >25 -2 MUCOUS PRESENT +1
- 20. . Reject if – score <0 Only by squamous epithelium Reject :->10 sq. epi/LPF
- 21. . To compare with culture result Wide diversity of bacterial forms – salivary contamination Predominance of 1 potential pathogenic form Gram pos. diplococci pneumococci Small slender GNB haemophilli Cluster GPC Staph. aureus
- 24. .Alveolar macrophages are recognized under low power by their characteristic staining reaction and their size. They are very densely staining and sometimes have a characteristic orange coloration. They are smaller than squamous epithelial cells but are larger than polymorphs. They are often found embedded in mucus strands. The presence of alveolar macrophages in a sputum smear indicates that the material under examination originated from the lower respiratory tract.
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- 32. Z-N STAIN Done on request GRADING IF THE SLIDE HAS RESUL T GRADING NO. OF FIELDS TO BE EXAMINED > 10 AFB/OIL FIELD POS 3+ 20 1 – 10 AFB/OIL FIELD POS 2+ 50 10 – 99 AFB/100 OIL FIELD POS 1+ 100 1 – 9 AFB/ 100 OIL FIELD POS SCANTY* 200NO AFB IN 100 OIL FIELD NEG 100
- 33. CRYPTOSPORIDIUM
- 34. AFB
- 35. THROAT SWAB GRAMS STAIN For cause of pharyngitis it is unreliable Charecteristic pattern of fusiform @ spirochetes can be seen:- vincent’s angina. Albert’s stain done only when requested
- 36. NASOPHARYNGEAL SWAB Done only in special cases Whooping cough – DFA
- 37. PUS GRAM’S STAIN Presence of relative number of polymorphs and bacteria WET FILM Presence of fungal elements Presence of motile bacteria Any refractile objects Spirochetes in pr. Syphilis by darkground microscopy Z-N STAIN When requested
- 38. PUS SPECIMEN
- 40. WET FILM •Microscopically observation of well mixed uncentrifuged fluid in a slide counting chamber. •Relative numbers of polymorphs and lymphocytes should be noted •If only slight contamination with blood ,leucocytes and erythrocytes to be counted separately.
- 41. •Leucocytes in numbers in excess of 1 per 1000 erythrocytes, suggest presence of meningitis. •0.2 mL •Cell count is performed in Fuchs-Rosenthal slide chamber
- 42. Counting fluid contains: •Glacial acetic acid 1ml •Propan 1,2diol 2.5ml •Crystal violet 1% in water 1.5 ml •Distilled water 100ml
- 43. GRAM STAIN Reaction to gram’s stain Morphology of organism Type of leucocyte Pus cells- Pyogenic bacterial meningitis Amoebic meningo-encephalitis
- 44. •BACTERIA •Gram-negative intracellular diplococci- Neisseria meningitides •Gram-positive diplococci or short streptococci Streptococcus pneumonia •Gram-negative bacilli- Haemophilus influenza, E.coli or other coliforms •Yeast cells unevenly stained ,irregular in size, showing budding- Cryptococcus neoformans
- 45. . INDIAN INK PREPARATION If yeast cell seen From the deposit Round to oval yeasts with clear halo- C.neoformans
- 46. Z-N STAIN:- •If requested • diagnosis of tuberculous meningitis is suspected WET FILM:- • for amoebic trophozoites •Acanthamoeba •Naegleria
- 47. GRAM STAIN OF CSF N.meningitidis:intracellular,gram-negative diplococci
- 48. GRAM STAIN OF CSF:S.pneumoniae:gram positive diplococci
- 49. Gram stain of CSF:H.influenzae:gram-negative pleomorphic coccobacilli
- 50. HIGH VAGINAL SWAB WET FILM For motile trichomonas vaginalis:- pear shaped with jerky motility Presence of polymorph Fungal elements:- hyphal form of candida GRAM STAIN Presence of pseudomycelium + yeast cells Clue cells GPB – Lactobacilli GN or gram variable bacilli AMSELS criteria
- 53. NUGENT SCORING Score Lactobacillus Gardnerella Curved bacteria Morphotype /Vision field Morphotype/Vision field Morpho/field 0 >30 0 0 1 5—30 <1 1—5 2 1—4 1—4 >5 3 <1 5—30 4 0 >30 Scores 0-3= Normal flora 4-6= Intermediate flora 7-10= BV
- 55. OCULAR SPECIMEN GRAM’S STAIN Type of predominant wbc Multinucleated epithelial cells- herpes infection WET FILM Motile trophozoites- acanthamoeba Fungal hyphae SPECIAL STAINS DFA stain- chlamydia - VZV,HSV
- 56. FUSARIUM
- 57. STOOL Routine smear examination not done Trophozoites are usually found in liquid or soft stools Cysts usually found in fully formed stool Should be examined within 30 min. of passage PVA slides can be made to store For enterobius-NIH swab Stool after saline spurge
- 58. WET FILMS Motility of any trophozoite Cyst STAINED PREPARATIONS IODINE WET MOUNT Detection of cysts Dead specimen of trophozoites For study of nuclear character for identification of species
- 59. HELMINTHIC EGGS
- 61. . HEIDENHAIN’S IRON- HEMATOXYLIN WHEATLEY’S TRICHROME METHOD E.histolytica cyst– Blue-green tinged with purple in green background SCOTCH – TAPE PREPARATION
- 64. .. STOLL’S METHOD OF EGG COUNTING: 4gm of stool are mixed with 56ml of N/10 NaOH in a thick glass tube and thoroughly mixed to make a uniform suspension. This is facilitated by adding a few glass beads and closing the mouth with a rubber stopper and then shaking vigorously. Exactly 0.15 ml of the emulsion is removed by a measuring pipette and placed on a large slide(3”x2”size).
- 65. . A coverslip measuring 22/40 mm is then put over it . With the help of a mechanical stage , all the eggs in the preparation are counted The number of eggs per gm of stool is obtained by multiplying the count of two such preparation by 100.
- 66. BLOOD PROTOZOAL INFECTIONS Malaria Babesiosis Chaga’s disease African Trypanosomiasis Leishmeniasis
- 67. HELMINTHIC INFECTION Lymphatic filariasis loasis
- 68. WET FILM Examined microscopically for large, motile, exo- erythrocytic parasites. Used for trypanosomes : rapid undulating and twisting movement microfilariae. : whip like motion Blood must be examined within 1 hour of collection to avoid morphological changes.
- 69. . For good parasite morphology, smears prepared within 1 hour. After that time, Schüffner's stippling and other dots may not be visible on stained films. However, overall organism morphology should still be excellent if smears are prepared within two hours.
- 70. PBS .
- 71. . STAINED SMEAR LEISH MAN’S STAIN GIEMSA STAIN JSB STAIN FIELD’S STAIN Thin film Thick film Contact method Puddle method : when blood with anti-coagulent used
- 72. . Blood films must be carefully reviewed for presence of any blood parasites. These may vary in size from small intraerythrocytic malaria or Babesia rings to large microfilariae. For this reason, it is recommended that blood films be scanned using a 10x or 20x objective to search for large forms (microfilariae, schizonts, gametocytes). This should be followed by a thorough examination (at least 300 fields), using the 100x oil immersion objective
- 74. . Malarial infections should be reported as the percentage of infected RBCs per 100 RBCs counted. At least 10 fields in thin film should be counted. In cases of low parasitemia (<5% parasitemia),number of parasites/100 WBCs is done. This count may be performed on either thick or thin blood films.
- 78. HOW RBC’S ARE SEEN RBCs – pale bluish-gray. Cytoplasm may contain pink stippling, called Schüffner's dots, in Plasmodium vivax. Small, pink-to-red rod-like inclusions, called “Maurer's clefts,” as in P. falciparum infected erythrocytes. Small blue specks in cytoplasm (or homogeneous bluish tinge) are found in reticulocytes, immature erythrocytes, and may be present in uninfected or infected erythrocytes.
- 79. WBC’S . Neutrophils, Lymphocytes, and Monocytes Pale blue-grey cytoplasm and purple nucleus. Eosinophils Coarse, pink granules in cytoplasm. Basophils Coarse, deep blue granules in cytoplasm.
- 80. ESTIMATION OF PARASITE DENSITY (NVBDCP) Number of parasites X 8000 ---------------------------------- = parasites per microlitre Number of leukocytes Simpler method of counting parasites in thick blood film. + = 1-10 parasites /100 (thick film) fields + + = 11-100 parasites /100 (thick film) fields + + + = 1-10 parasites /single (thick film) fields + + + + = >10 parasites /single (thick film) field
- 81. MICROFILARIA COUNT With help of a haemoglobinometer pipette 20 mm square of blood is placed on a clean glass slide. Dried as thick film, dehaemoglobinised and stained in the usual manner. Total number of microfilariae in thick smear multiplied by 50 will give the number per ml of blood.
- 84. LEISHMANIA Amastigotes of L. donovani (LD bodies) are seen in peripheral blood smear. Cytoplasm – pale blue Nucleus – red Parabasal body – deep red Kinetoplast – bright red
- 85. FLUORESCENT MICROSCOPY Kawamoto acridine orange process Use of benzothiocarboxypurine (BCP) Used when parasitic count >100/ml
- 86. HAIR,SKIN,NAIL SCRAPPING First proof of the etiology of disease Traditionally KOH preparation Calcofluor white stain- Inherent weak fluorescence when viewed in blue light MALDI-TOF
- 87. 20% KOH + 40% Aq. DMSO(DIMETHYL SULPHOXIDE) 20% KOH + CALCOFLUOR Adhesive tape strippings Short curved hyphae with group of spherical cells Bananas and grapes Spaghetti and meat balls MALASSEZIA FURFUR
- 88. DERMATOPHYTES
- 89. .