The document provides information about the history and types of microscopy. It discusses early microscopes developed by Hans Lippershey, Hans Janssen, and Giovanni Faber in the 1590s. Antoni van Leeuwenhoek is noted for his discoveries of micro-organisms in the 1670s using microscopy. In 1893, August Köhler developed Köhler illumination, important for modern light microscopy. Light microscopes and electron microscopes are described. Microscopic examination of samples can provide diagnostic information for conditions like malaria and other infectious diseases.
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
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
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.
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.
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
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
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
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
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
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.
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.
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.
75.
76.
77.
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.
82.
83.
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
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
