1 UNIT 3 COLLECTION AND PROCESSING OF SAMPLE FOR FUNGAL INFECTION IN SKIN, NAIL AND HAIR LEARNING OBJECTIVE To understand the concept of skin fungal infection. To understand the concept of nail fungal infection. To understand the concept of hair fungal infection. To understand the concept of LCB. To understand the concept of KOH. To understand the concept of INDIA INK. 3.1 COLLECTION OF SKIN SCRAPPINGS Dermatophytes such as Trichophyton, Microsporum and Epidermophyton causes skin infection. It may be caused by skin infection. 3.1.1 PROCEDURE First of all clean the lesion with 70% ethanol. Scrap the material from the periphery of the lesion with a sterilized scalpel.. Place the material in a clean and sterilized petridish . The scrapping specimen should be transported to the clinical lab in petridish.. If 70% ethanol causes burning sensation into the lesion then it should be cleaned with distilled water. 3.2 COLLECTION OF NAIL SCRAPPINGS Aspergillus, Candida and Dermatophytes like Trichophyton, Microsporum, Epidermophyton causes nail infection. 3.2.1 PROCEDURE First of all clean the nails with 70% ethanol. Scrap the material from the distal end of the nail with a sterilized scalpel. Discard the first 4-5 scrappings and collect the latter ones. Place the material in a clean petridish. The scrapped specimen should be transportd to the clinical laboratory. 3.3 COLLECTION OF HAIR SCRAPPINGS Piedra, Trichosporon, Trichophyton, Microsporum and Epidermophyton causes fungal infection in hair. Infected hair is identified by exposure to uv light. 3.3.1 PROCEDURE Hair should be collected from areas of scaling or alopecia. The infected hair are plucked by using sterilized forceps. In young patients, removal of hair is difficultso place a strip of clear tape over the infected area and then remove it.
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1
UNIT 3
COLLECTION AND PROCESSING OF SAMPLE FOR FUNGAL INFECTION IN SKIN,
NAIL AND HAIR
LEARNING OBJECTIVE
To understand the concept of skin fungal infection.
To understand the concept of nail fungal infection.
To understand the concept of hair fungal infection.
To understand the concept of LCB.
To understand the concept of KOH.
To understand the concept of INDIA INK.
3.1 COLLECTION OF SKIN SCRAPPINGS
Dermatophytes such as Trichophyton, Microsporum and Epidermophyton causes skin
infection. It may be caused by skin infection.
3.1.1 PROCEDURE
First of all clean the lesion with 70% ethanol.
Scrap the material from the periphery of the lesion with a sterilized scalpel..
Place the material in a clean and sterilized petridish .
The scrapping specimen should be transported to the clinical lab in petridish..
If 70% ethanol causes burning sensation into the lesion then it should be cleaned with
distilled water.
3.2 COLLECTION OF NAIL SCRAPPINGS
Aspergillus, Candida and Dermatophytes like Trichophyton, Microsporum,
Epidermophyton causes nail infection.
3.2.1 PROCEDURE
First of all clean the nails with 70% ethanol.
Scrap the material from the distal end of the nail with a sterilized scalpel.
Discard the first 4-5 scrappings and collect the latter ones.
Place the material in a clean petridish.
The scrapped specimen should be transportd to the clinical laboratory.
3.3 COLLECTION OF HAIR SCRAPPINGS
Piedra, Trichosporon, Trichophyton, Microsporum and Epidermophyton causes
fungal infection in hair. Infected hair is identified by exposure to uv light.
3.3.1 PROCEDURE
Hair should be collected from areas of scaling or alopecia.
The infected hair are plucked by using sterilized forceps.
In young patients, removal of hair is difficultso place a strip of clear tape over the
infected area and then remove it.
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Place the collected hair into a clean and sterilized petridish.
The specimen should be transported to the clinical laboratory in petridish.
3.4 POTASSIUM HYDROXIDE PREPARATION
Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal elements in
clinical specimen, as it clears the specimen making fungal elements more visible during
direct microscopic examination. KOH is a strong alkali. When specimen such as skin, hair,
nails or sputum is mixed with 20% w/v KOH (preparation of KOH is posted at the end of this
post), it softens, digests and clears the tissues (e.g., keratin present in skins) surrounding the
fungi so that the hyphae and conidia (spores) of fungi can be seen under microscope.
3.4.1USES
In diagnostic laboratories, KOH mount is one of the main methods of investigating fungal
infections. It is used as a primary screening tool, it detects fungal elements present but may
not necessarily identify the species of the fungi. To identify the fungal isolate, specimen must
be cultured in either general purpose fungal culture media such as Sabouraud Dextrose Agar
(SDA) or specific media based on the type of anticipated isolate.) KOH preparation is
recommended in the following suspected conditions (this is not the exclusive lists);
3.4.2 PROCEDURE OF KOH PREPARATION
Place a drop of KOH solution on a slide.
Transfer the specimen (small pieces) to the drop of KOH, and cover with glass. Place the slide in a petri dish, or other container with a lid, together with a damp piece of
filter paper or cotton wool to prevent the preparation from drying out.
As soon as the specimen has cleared, examine it microscopically using the 10X and 40X objectives with the condenser iris diaphragm closed sufficiently to give a good
contrast.
If too intense a light source is used the contrast will not be adequate and the
unstained fungi will not be seen.
3.4.3 DISADVANTAGES OF KOH PREPARATION
Experience required since background artifacts are often confusing.
Clearing of some specimens may require an extended time
3.4.4PROCEDURE TO MAKE KOH PREPARATION
Weigh 20 g potassium hydroxide (KOH) pellets.
Transfer the chemical to a screw-cap bottle.
Add 50 ml distilled water, and mix until the chemical is completely dissolved, add remaining distilled water and make the volume 100 ml.
Label the bottle and mark it corrosive. Store it at room temperature. The reagent is
The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method
of staining and observing fungi and is simple to prepare. The preparation has three
components:
Phenol: kills any live organisms;
Lactic acid : It preserves fungal structures, and
Cotton blue : It stains the chitin in the fungal cell walls.
3.5.1PREPARATION
Place a drop of seventy percent alcohol on a clean microscope slide.
Material from cultures of filamentous fungi should be removed using a stiff
inoculating wire not the loop used for manipulations with bacteria or yeasts.
Flame the wire by holding it upright in the hottest part of the Bunsen flame, just
above the blue cone, until the whole length of the wire glows red hot.
You must ensure that the inoculating wire has cooled before placing it in a fungal
culture – it should have cooled sufficiently after approximately ten seconds.
Remove the cap from the tube but do not put it on the bench. Kill any contaminating microorganisms by flaming the neck of the tube.
Remove a small amount of the culture. For fungal cultures, it is often useful to take a little of the agar medium together with the fungus. In any case, the material should be
disturbed as little as possible when being transferred to the slide.
Flame the neck of the tube once more and replace the cap.
Immerse the fungal material in the drop of seventy percent alcohol. This drives out the air trapped between the hyphae.
Tease out the material very gently with mounted needles.
Do not forget to sterilise the inoculating wire and the needles after use by heating to red heat in a Bunsen flame
Fungal structures are readily visualised after staining with a lactophenol cotton blue dye preparation.
Before the alcohol dries out add one or at most two drops of the stain. A common fault is to add too much to the preparation. Holding the coverslip between your index
finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is now ready for examination.
Make the initial examination using a low power objective lens. The thinner parts of the preparation, generally around the edges of the mounted material, will yield the
best images.
Switch to a higher power 40X objective for more detailed examination of spores.
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3.6 INDIA INK
The main purpose of Negative staining is to study the morphological shape,
size and arrangement of the bacteria cells that is difficult to stain.
eg: Spirilla. It can also be used to stain cells that are too delicate to be heat-
fixed.It is also used to prepare biological samples for electron microscopy.
It is used to view viruses, bacteria, bacterial flagella, biological membrane
structures and proteins or protein aggregates, which all have a low electron-
scattering power. It is also used for the study and identification of aqueous
lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M)
and inverted hexagonal HII cylindrical (H) phases by Negative staining
transmission electron microscopy.The main purpose of Negative staining is to
study the morphological shape, size and arrangement of the bacteria cells that
is difficult to stain. eg: Spirilla. It can also be used to stain cells that are too
delicate to be heat-fixed. It is also used to prepare biological samples for
electron microscopy. It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all
have a low electron-scattering power. It is also used for the study and
identification of aqueous lipid aggregates like lamellar liposomes (le),
inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H)
phases by Negative staining transmission electron microscopy.
VERY SHORT ANSWER TYPE QUESTIONS
Q1. Expand LCB.
Q2 Expand KOH.
Q3 Define negative staining.
Q4. Use of cotton blue.
Q5. Use of lactic acid.
SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS
Q1.Discuss the principle and procedure of LCB.
Q2. Discuss the principle and procedure of KOH
. Q3. Discuss the principle and procedure of India ink.
Q4.Discuss collection and processing of skin specimen.
Q5. Discuss collection and processing of skin specimen
Q6. . Discuss collection and processing of skin specimen
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UNIT4
FUNGAL CULTIVATION
LEARNING OBJECTIVE
To learn the concept of fungal cultivation.
To learn the concept of isolation of candida
To learn the concept of of isolation of Dermatophytes.
To learn the concept of of isolation of pencillium
To learn the concept of of isolation of Rhizopus
To learn the concept of of isolation of Mucor
To learn the concept of of isolation of Aspergillus.
4.1 TECHNIQUES OF FUNGAL CULTIVATION
4.1.1 INTRODUCTION
Aseptic Technique: In most microbiological procedures, it is necessary to protect
instruments, containers, media, and stock cultures from contamination by
microorganisms constantly present in the environment. Aseptic technique involves the
sterilization of tools, glassware, and media before use as well as measures to prevent
subsequent contamination by contact with nonsterile objects.
Equipment and Work Area: To culture bacteria or fungi, you need the
following materials
Disinfectant solution such as 70% ethanol, 4% household bleach solution.
Alcohol or gas (Bunsen) burner.
Inoculating loop for bacteria, yeasts, and fungi with abundant spores; scalpel
or half-spearpoint needle for other fungi Stock culture (the original culture
from which other cultures will be started)
Sterile medium in petri dishes or culture tubes
Soap for washing hands.
Lab coat or old, clean shirt, especially while you are staining cultures.
Before working with bacterial or fungal cultures, always wash your hands
with soap and water. Next, prepare a work area. Select an area that is as free
from drafts as possible. Turn off the air-conditioner and fans, and close all
windows and doors. Wipe the work area with 70% ethanol or a similar
disinfectant solution. Arrange your materials conveniently on the clean work
surface. Do not smoke, eat, or drink while working with cultures. Media
Preparation The first step in media preparation is to assemble the equipment
and ingredients. You will need a balance, spatula, weighing paper, 1-L
graduated cylinder, glass stirring rod, a large flask or beaker, and culture
tubes or petri dishes. You can either use a recipe to prepare a particular
medium from scratch or purchase any of the commercially available
dehydrated media. The media most commonly used are nutrient agar
(bacteria), potato dextrose agar (fungi), and Sabouraud dextrose agar (fungi).
After assembling the equipment and ingredients, weigh the dry ingredients
accurately. Place a sheet of weighing paper on the pan to protect the balance
and to facilitate transferring the material into a flask. Using the weighing
paper as a funnel, pour the dry ingredients into a large flask or beaker. Add
the proper amount of distilled water and swirl the flask to dissolve the dry
material. Agar-containing media must be heated slowly, just to boiling, to
dissolve the agar. Gently agitate the medium during the heating process by
either stirring or shaking the flask. Watch the flask carefully: agar burns
easily and boils over quickly. Pour the liquid agar or broth into bottles or
culture tubes and cap them loosely. Autoclave the medium in the bottles or
culture tubes to sterilize it. If the medium is to be used to pour dishes,
autoclave it in the plugged flask in which it was mixed. When sterilization is
complete, lay the tubes on a slant tray. Tighten the tops for storage only after
the agar solidifies. If plates are to be poured, disinfect the work area and stop
all air drafts. Let the flask cool until it is easy to handle (20 to 40 minutes).
Lay out sterile petri dishes and light a Bunsen burner. Remove the stopper
from the flask and flame the mouth. Lift the cover of the dish at just enough of
an angle to pour in the medium. Pour the agar slowly to avoid bubble
formation; if bubbles do form, pass the burner flame quickly over the surface
of the agar severa times, which should cause the bubbles to burst. Pour
enough agar to fill the dish about one-half full, replace the cover, and allow
the dish to stand undisturbed until the agar solidifies.
Sterilization Procedures Many microorganisms produce highly resistant
spores that remain viable even after exposure to dry heat or boiling water for
several hours. Steam under pressure is used to increase the temperature
enough to kill any contaminating microorganisms. Steam penetrates
wrappings and loosely capped articles, sterilizing the contents. The home
pressure cooker works on this principle. An autoclave is, in essence, a large,
self-contained pressure cooker that goes through the heating, sterilizing, and
cooling cycles automatically. If an autoclave is not available, you can use a
large pressure cooker on a stove as long as you follow a few rules:
Read the directions for your brand of cooker and follow them
carefully.
Make sure there is sufficient water in the cooker.
Don’t start timing until 15 pounds per square inch (psi) have been
reached. 4. At the end of 15 minutes, allow the pressure cooker to cool
slowly. Media should be sterilized for 15 minutes at a temperature of
121°C and a pressure of 15 psi. Glassware and contaminated articles
like old stock cultures should be autoclaved for 30 minutes at 121°C
and 15 psi.
4.2 GENERAL CHARACTERISTICS AND ISOLATION OF
DERMATOPHYTES
Superficial fungal infections of the skin is public health problem .
Dermatophytosis which involves keranized tissues such as superficial skin,
nails and hair, is the commonest condition that is encountered in the
dermatology clinics. These infections are usually ignored and patient presents
to outpatient department only if cosmetically disfiguring or due to
inconvenience caused by pruritus. This results in many not seeking medical
help immediately thus increasing the disease load in the community and
increased risk of transmission. The epidemiology of dermatophytosis has
evolved over the last many decades. Whether developed countries or
developing countries these changes have occurred due to changing life styles,
economy, increasing leisure activities and environmental factors. People who
are exposed to soil, and animals occupationally or as a leisure activity, are at
risk of acquiring dermatophyte infectionof nail, it is left for longer period till
the cells are dissolved. The slide was examined for the presence of fungal
elements.
Culture: Sabouraud’s dextrose agar (SDA) with gentamicin (8mg/litre)were
used for culturing the specimens initially. Later dermatophyte test
medium(DTM) was also included for culturing the specimens. All cultures
were incubated at 250 C in biochemical oxygen demand incuators/B.O.D
incubator for 4 weeks before issuing a negative report. Plates and tubes were
examined everyday during first week and every two days thereafter. This was
done to detect any bacterial or saprophytic fungal contamination and also to
re-inoculate in case contamination occurred. The day of the appearance of the
suspected dermatophyte colony was noted for assessing the rate of growth of
the isolates.
4.3 GENERAL CHARACTERISTICS AND ISOLATION OF
PENICILLIUM.
4.3.1MACROSCOPIC APPEARANCE
Surface: Texture velvety to powdery; Green, blue-green, gray-green, white,
yellow, or pinkish on the surface.
Reverse: Usually white to yellowish, sometimes red or brown.
Growth Rate: Moderately rapid to rapid.
Note: If the isolate produces a red reverse and diffuse pigment in the agar, P. marneffei must be
considered and the organism should be tested for thermal dimorphism; this is especially relevant if
the patient has recently visited southeast Asia.
4.3.2 DISTINGUISHING FEATURES
Penicillium is distinguished by its frequently greenish colonies and its branching or simple
conidiophores supporting phialides in brush-like clusters known as penicilli. It is
differentiated from Paecilomyces by its phialides lacking long, pointed apical extensions. In
contrast to Scopulariopsis , its conidia lack a truncate base. P. marneffei produces downy
gray-green colonies, often with a brownish or red tint caused by the presence of red or
yellow pigmented sterile hyphae in the colony. The colonies, when incubated at 37 degrees C.
on Sabouraud Dextrose Agar, characteristically lose this pigmentation and convert into
yeast-like cells multiplying by fission. The diagnosis of infection due to P. marneffeirests on
the histopathologic demonstration of cells multiplying by fission in the interior of leukocytes.
4.3.3 HABITAT
Penicillium are cosmopolitan, predominant in regions of temperate climate. Penicillia figure
among the most common types of fungi isolated form the environment. Of the approximately
150 recognized species, some are frequently implicated in the deterioration of food products
where they may produce mycotoxins. Other species are producers of penicillin. Infections
with P. marneffei are primarily acquired in mountainous provinces of Northern Thailand,
Laos, Myanmar, and Southeastern China.
4.3. 4 PATHOGENICITY
Normally considered nonpathogenic with the exception of Penicillium marneffei , a
dimorphic species capable of causing infection of the lymphatic system, the lungs, the liver,
the skin, the spleen, and the bones. It has been known to cause keratitis (inflammation of the
cornea), external ear, respiratory, and urinary tract infections, and endocarditis after
insertion of valve prostheses.
4.3.5 RECOMMENDED MEDIA
Corn Meal Agar
Inhibitory Mold Agar
Malt Extract Agar
Potato Dextrose Agar
Sabouraud Dextrose Agar
Wort Agar
Incubate at 25 degrees C. for 2-7 days.
4.4 GENERAL CHARACTERISTICS AND ISOLATION OF RHIZOPUS
4.4.1MICROSCOPIC APPEARANCE
Hyphae broad, not or scarcely septate; rhizoids and stolons present; sporangiophores brown,
solitary or in tufts on the stolons, diverging from the point at which the rhizoids form;
sporangia rather round; apophysis absent or scarcely apparent; sporangiophores ovoid.
4.4.2 MACROSCOPIC APPEARANCE
Surface: Texture deeply cottony; White becoming gray-brown on surface.
Reverse: Pale white.
Growth Rate: Very rapid growth.
4.4.3 DISTINGUISHING FEATURES
Rhizopus is recognized by the presence of well developed rhizoids situated at the point where
sporangiophores are attached to the stolons. In contrast to Mucor , Rhizomucor and Absidia ,
4.4.4 HABITAT
Rhizopus are cosmopolitan, frequently isolated from soil and agricultural products (cereal,
vegetables, etc.). Certain species are plant pathogens.
Pathogenic
Maximum
Growth
Temperature
Rhizoid
Length
(um)
Sporaniophore
Length
(um)
Sporangium
Length
(um) Columellae
Sporangio-
spores
Positive 50-52°C 100-
120
200-1000 40-130 Slightly
elongated;
distinct
apophysis
Equal size,
average length 4-
6um; smooth to
slightly striated;
almost round to
slightly elongated
Postive 40-46°C 150-
300
500-3500 50-250 Almost
round
Variable size,
average length 6-
8um; striated;
elongated to
lemon-shaped
Negative 30-32°C 300-
350
1500-4000 150-350 Almost
round
Variable in size,
average length 9-
11um; very
striated;polyhedric
4.4.5PATHOGENICITY
Rhizopus is the principal agent of mucormycosis (formally zygomycosis). This rapidly
progressing infection is characterized by the cerosis of tissues and the production of infarcts
in the brain, the lungs, and the intestines. Primarily, it is patients suffering from diabetic
ketoacidosis, malnutrition, severe burns, or immunocompromising conditions who are most
at risk to develop this type of infection.
4.4.6 RECOMMENDED MEDIA
Corn Meal Agar
Malt Extract Agar
Potato Dextrose Agar
Sabouraud Dextrose Agar
Wort Agar
INCUBATION
Temperature: 25 degrees C.
Time: 2-7 days.
4.5GENERAL CHARACTERISTICS AND ISOLATION OF MUCOR
Mucor is a filamentous fungus found in soil, plants, decaying fruits and vegetables. As well
as being ubiquitous in nature and a common laboratory contaminant, Mucor spp. may cause
infections in man, frogs, amphibians, cattle, and swine. Most of the Mucor spp. are unable to
grow at 37°C and the strains isolated from human infections are usually one of the few
thermotolerant Mucor spp. The genus Mucor contains several species. The most common
Q5. What are the characteristics of antigen-antibody reactions.
UNIT 9
SEROLOGICAL TESTS
LEARNING OBJECTIVE:
To learn the concept of widal test.
To learn the concept of Antistreptolysin O test.
To learn the concept of C- Reactive protein test.
To learn the concept of Rheumatoid factor.
To learn the concept of VDRL
To learn the concept of ELISA
9.1 WIDAL TEST
9.1.1 INTRODUCTION
Widal Test is an agglutination test which detects the presence of serum agglutinins (H
and O) in patients serum with typhoid and paratyphoid fever.
When facilities for culturing are not available, the Widal test is the reliable and can
be of value in the diagnosis of typhoid fevers in endemic areas.
It was developed by Georges Ferdinand Widal in 1896.
The patient’s serum is tested for O and H antibodies (agglutinins) against the
following antigen suspensions (usually stained suspensions):
S. Typhi 0 antigen suspension, 9, 12
S. Typhi H antigen suspension, d
S. Paratyphi A 0 antigen suspension, 1, 2, 12
S. Paratyphi A H antigen suspension, a
S. Paratyphi B 0 antigen suspension, 1, 4, 5, 12
S. Paratyphi B H antigen suspension, b, phase 1
S. Paratyphi C 0 antigen suspension, 6, 7
S. Paratyphi C H antigen suspension, c, phase 1
Salmonella antibody starts appearing in serum at the end of first week and rise
sharply during the 3rd week of endemic fever. In acute typhoid fever, O agglutinins
can usually be detected 6–8 days after the onset of fever and H agglutinins after 10–
12 days.
It is preferable to test two specimens of sera at an interval of 7 to 10 days to
demonstrate a rising antibody titre.
Salmonella antigen suspensions can be used as slide and tube techniques.
9.1.2 PRINCIPLE OF WIDAL TEST
Bacterial suspension which carry antigen will agglutinate on exposure to antibodies
to Salmonellaorganisms. Patients’ suffering from enteric fever would possess antibodies in
their sera which can react and agglutinate serial doubling dilutions of killed,
coloured Salmonella antigens in a agglutination test.
The main principle of widal test is that if homologous antibody is present in patients serum, it
will react with respective antigen in the reagent and gives visible clumping on the test card
and agglutination in the tube. The antigens used in the test are “H” and “O” antigens
of Salmonella Typhi and “H” antigen of S.Paratyphi. The paratyphoid “O” antigen are not
employed as they cross react with typhoid “O” antigen due to the sharing of factor 12. “O”
antigen is a somatic antigen and “H” antigen is flagellar antigen.
9.1.3 PREPARATION
H suspension of bacteria is prepared by adding 0.1 per cent formalin to a 24 hours
broth culture or saline suspension of an agar culture.
For preparation of O suspensions of bacteria, the organisms is cultured on phenol
agar (1:800) to inhibit flagella.
Standard smooth strains of the organism are used; S Typhi 901, O and H strains are
employed for this purpose.
The growth is then emulsified in small volume of saline, mixed with 20 times its
volume of alcohol, heated at 40° C to 50° C for 30 minutes and centrifuged.
The antigens are treated with chloroform (preservative) and appropriate dyes are
added for easy identification of antigens.
9.1.4. PROCEDURE
o Place one drop of positive control on one reaction circles of the slide.
o Pipette one drop of Isotonic saline on the next reaction cirlcle. (-ve Control).
o Pipette one drop of the patient serum tobe tested onto the remaining four
reaction circles.
o Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction
circles. (PC & NC).
o Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four
reaction circles.
o Mix contents of each circle uniformly over the entire circle with separate
mixing sticks.
o Rock the slide, gently back and forth and observe for agglutination
macroscopically within one minute.
9.1.5 INTERPRETATION OF TEST RESULTS
Agglutination is a positive test result and if the positive reaction is observed with 20 ul of test
sample, it indicates presence of clinically significant levels of the corresponding antibody in
the patient serum. No agglutination is a negative test result and indicates absence of
clinically significant levels of the corresponding antibody in the patient serum.
9.2 ANTISTREPTOLYSIN O TEST
9.2.1 INTRODUCTION
It is a rapid latex agglutination test for the qualitative and semi-quantitative determination of
anti-streptolysin-O antibodies (ASO) in serum. In infections caused by β-haemolytic
streptococci, streptolysin-O is one of the two hemolytic exotoxins liberated from the
bacteria that stimulates production of ASO antibodies in the human serum. The presence
and the level of these antibodies in a serum may reflect the nature and severity of infection.
ASO latex reagent is a stabilized buffered suspension of polystyrene latex particles that have
been coated with Streptolysin O.
9.2.2 MATERIALS USED
ASO Antigen: A stabilized buffered suspension of polystyrene latex particles coated with Streptolysin O and 0.1% sodium azide as preservative. Shake well prior to use.
ASO Positive Control: Human serum containing more than 200 IU/ml ASO and 0.1%
sodium azide as preservative.
ASO Negative Control: Human serum containing 0.1% sodium azide as preservative.
Sufficient disposable pipettes.
Glass test slide.
9.2.3 PROCEDURE
Bring all test reagents and samples to room temperature.
Use a disposable pipette to draw up and place one free-falling drop of each undiluted
sample into its identified circle of the slide. Retain each pipette for mixing in step 5.
Deliver one free-falling drop of positive and negative control into its identified circle.
Mix the ASO latex reagent by gently shaking. Add one free-falling drop of reagent to
each control and sample.
Using the flattened end of the appropriate plastic pipette as a stirrer (step 2),
thoroughly mix each sample with reagent within the full area of the circle.
Discard the disposable pipette.
Slowly rock the slide for exactly two (2) minutes and observe for agglutination under a high intensity light.
Record results.
Re-wash glass slide for future use
9.2.4 TEST RESULT
A test sample is considered to contain ASO antibodies in excess of 200 IU/ml when
agglutination (clumping) is observed when compared to the result of the negative control.
9.3 C-REACTIVE PROTEIN TEST
9.3.1 INTRODUCTION
Rheumatoid Factors are autoantibodies that react with individuals own immunoglobulin.
These antibodies are usually directed against the Fc fragment of the human IgG. RF have
been associated with three major immunoglobulin classes: IgM, IgG, and IgA. Of these IgM
and IgG are the most common. The formation of immune complex in the joint space leads to
the activation of complement and destructive inflammation, causing rheumatoid arthritis
(RA).
As indicated by its name, RF has particular application to diagnosis and monitoring of
rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting
primarily the joints and periarticular tissues. Rheumatoid factor is detected in 60-80% of
cases of diagnosed rheumatoid arthritis. However, it is also detectable sometimes in the
serum of patients with Systemic Lupus Erythematosus (SLE) and in certain non-rheumatic
conditions. Elevated values may also be observed in normal elderly population.
9.3.2 PRINCIPLE
A number of methods are available for testing of RF. The most commonly used serological
method is based on latex agglutination test. As RF is an IgM class of antibody directed
against the Fc portion of the IgG molecule, it is detected by it’s ability to agglutinate
the latex. Reagent used is a suspension of polystyrene latex particles in glycine-saline buffer.
9.3.3 PROCEDURE
Bring all reagents and specimens to room temperature.
Place one drop of the positive control and 40ul of the patient serum into separate circles on the slide.
Gently and add one drop of RF latex reagent on each circle of sample to be tested and
control.
Use separate Applicator sticks/stir sticks to spread reaction mixture over entire area of the particular field.
Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates slowly.
Observe for agglutination after 2 minutes under bright artificial light.
9.3.4 INTERPRETATION
Agglutination of latex particles is considered a positive reaction, indicating the presence of
rheumatoid factor at a significant and detectable level.
Positive result: An agglutination of the latex particles suspension will occur within two
minutes, indicating a RF level of more than 18 IU/ml.
Negative result: No agglutination of the latex particles suspension within two minutes.
9.5 VENERAL DISEASE RESEARCH LABORATORY
9.5.1 INTRODUCTION
Venereal Disease Research Laboratory (VDRL) Test is a slide flocculation test
employed in the diagnosis of syphilis. Since the antigen used in this test is cardiolipin,
which is a lipoidal extracted from beef heart, it is not a specific test. This test is also
classified as non-specific or non-treponemal or standard test. The antibodies reacting
with cardiolipin antibodies have been traditionally (but incorrectly) termed “regain”.
Principle: Patients suffering from syphilis produce antibodies that react with cardiolipin
antigen in a slide flocculation test, which are read using a microscope. It is not known if
the antibodies that react with cardiolipin are produced against some lipid component of
Treponema pallidum or as a result of tissue injury following infection. Requirements:
Patient’s serum, water bath, freshly prepared cardiolipin antigen, VDRL slide,
mechanical rotator, pipettes, hypodermic syringe with unbeveled needle and
microscope. Known reactive and non-reactive serum controls are also required.
VDRL antigen: The cardiolipin antigen is an alcoholic solution composed of 0.03%
cardiolipin, 0.21% lecithin and 0.9% cholesterol. The cardiolipin antigen must be
freshly constituted each day of test. The working antigen is a buffered saline suspension
of cardiolipin.
VDRL slide: This is a glass slide measuring 2 X 3 inch with 12 concave depressions,
each measuring 16 mm in diameter and 1.78 mm deep.
9.5.2 PROCEDURE
Patients’ serum is inactivated by heating at 56o C for 30 minutes in a water bath to
remove non-specific inhibitors (such as complement)
The test can be performed both qualitatively and quantitatively.
Those tests that are reactive by qualitative test are subjected to quantitative test to
determine the antibody titres.
0.05 ml of inactivated serum is taken into one well.
1/60th ml (or 1 drop from 18 gauge needle) of the cardiolipin antigen is then added
with the help of a syringe (unbeveled) to the well and rotated at 180 rpm for 4
minutes.
Every test must be accompanied with known reactive and non-reactive controls.
The slide is then viewed under low power objective of a microscope for flocculation.
The reactive and non-reactive controls are looked first to verify the quality of the
antigen.
Depending on the size the results are graded as weakly reactive (W) or reactive (R).
Reactive samples are then subjected to quantitative test.
9.5.3 QUANTITATIVE TEST
This is performed to determine the antibody titres. The serum is doubly diluted in saline
from 1in 2 to 1:256 or more. 0.05 ml of each dilution is taken in the well and 1/60 ml of
antigen is added to each dilution and rotated in a rotator. The results are then checked
under the microscope. The highest dilution showing flocculation is considered as
reactive titre. Sometimes, due to very high level of antibodies in the serum (prozone
phenomenon) the qualitative test may be non-reactive. If the clinical findings are
strongly suggestive of syphilis, a quantitative test may be directly performed on the
serum specimen.
9.5.4 CSF VDRL
VDRL test may also be performed on CSF samples in the diagnosis of neurosyphilis.
Quantitative VDRL is the test of choice on CSF specimens. However, there are some
variations in this test.
The antigen is diluted in equal volumes with 10% saline, CSF must not be heated (or
inactivated), the volume of antigen solution taken is 0.01 ml (or 1 drop from 21 gauge
needle) and rotation time is 8 minutes.
Rest of the procedure remains same. Significance of VDRL test: VDRL test becomes
positive 1-2 weeks after appearance of (primary lesion) chancre. The test becomes
reactive (50-75%) in the late phase of primary syphilis, becomes highly reactive
(100%) in the secondary syphilis and reactivity decreases (75%) thereafter.
Treatment in the early stages of infection may completely suppress production of
antibodies and result in non-reactive tests. Effective treatment in the primary or
secondary stages results in rapid fall in titre and the test may turn non-reactive in few
months.
Treatment in latent or late syphilis has very little effect on the titre and the titres may
persist at low levels for long periods.
Since the titre falls with effective treatment, it can be used for assessment of prognosis.
VDRL test is more suitable as a screening agent than a diagnostic tool. VDRL test is
also helpful in the diagnosis of congenital syphilis. Since passively transferred
antibodies through placenta may give false reactive test in serum of the infant, a repeat
test after a month showing no increase in titre may help rule out congenital syphilis.
Since the test employs a non-treponemal antigen, there are many chances of false
positive results. False positivity (other than technical) may be due to physiological of
pathological conditions. These are called biological false positives (BFP). If the remain
positive for less than 6 months it is considered acute and they remain positive for longer
than 6 months it is called chronic BFP. The physiological reasons for BFP include
pregnancy, menstruation, repeated blood loss, vaccination, severe trauma etc while the
reasons for pathological BFP include malaria, infectious mononucleosis, hepatitis,
etc. A reactive VDRL test does not necessarily imply that the person is syphilitic. The
diagnosis must be made in conjunction with clinical findings. Any reactive VDRL test
must be confirmed with a specific or treponemal test such as TPHA, FTA-ABS test.
9.6 ELISA
9.6.1 INTRODUCTION
ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter Perlmann
and Eva Engvall at Stockholm University in Sweden. It is a common laboratory technique
which is usually used to measure the concentration of antibodies or antigens in blood.
ELISA is a plate based assay technique which is used for detecting and quantifying
substances such as peptides, proteins, antibodies and hormones. An enzyme conjugated with
an antibody reacts with colorless substrate to generate a colored product. Such substrate is
called chromogenic substrate. A number of enzymes have been used for ELISA such as
alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific substrate
such as ortho-phenyldiamine dihydrochloride (for peroxidase), paranitrophenyl phosphate
(for alkaline phosphatase) are used which are hydrolysed by above enzymes to give colored
end product.
9.6.2 PRINCIPLE
ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated in a
well, and each well contains a different serum. A positive control serum and a negative
control serum would be included among the 96 samples being tested. Antibodies or antigens
present in serum are captured by corresponding antigen or antibody coated on to the solid
surface. After some time, the plate is washed to remove serum and unbound antibodies or
antigens with a series of wash buffer. To detect the bound antibodies or antigens, a
secondary antibodies that are attached to an enzyme such as peroxidase or alkaline
phosphatase are added to each well. After an incubation period, the unbound secondary
antibodies are washed off. When a suitable substrate is added, the enzyme reacts with it to
produce a color. This color produced is measurable as a function or quantity of antigens or
antibodies present in the given sample. The intensity of color/ optical density is measured at
450nm. The intensity of the color gives an indication of the amount of antigen or antibody.
9.6.3TYPES OF ELISA
Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody
and Antigen.
Indirect ELISA
Sandwich ELISA
Competitive
9.6.4 INDIRECT ELISA
Antibody can be detected or quantitatively determined by indirect ELISA. In this technique,
antigen is coated on the microtiter well. Serum or some other sample containing primary
antibody is added to the microtiter well and allowed to react with the coated antigen. Any
free primary antibody is washed away and the bound antibody to the antigen is detected by
adding an enzyme conjugated secondary antibody that binds to the primary antibody.
Unbound secondary antibody is then washed away and a specific substrate for the enzyme is
added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end
product is measured by spectrophotometric plate readers that can measure the absorbance of
all the wells of 96-well plate.
9.6.5 PROCEDURE OF INDIRECT ELISA
Coat the micro titer plate wells with antigen.
Block all unbound sites to prevent false positive results.
Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c.
Wash the plate, so that unbound antibody is removed.
Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).
Wash the plate, so that unbound enzyme-linked antibodies are removed.
Add substrate which is converted by the enzyme to produce a colored product.
Reaction of a substrate with the enzyme to produce a colored product.
9.6.6 ADVANTAGES OF INDIRECT ELISA
Increased sensitivity, since more than one labeled antibody is bound per primary
antibody.
A wide variety of labeled secondary antibodies are available commercially.
Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Versatile because many primary antibodies can be made in one species and the same
labeled secondary antibody can be used for detection.
Flexibility, since different primary detection antibodies can be used with a single labeled
secondary antibody.
Cost savings, since fewer labeled antibodies are required.
Different visualization markers can be used with the same primary antibody.
9.6.7 DISADVANTAGES OF INDIRECT ELISA
Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
An extra incubation step is required in the procedure.
9.6.8 SANDWICH ELISA
Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the
microtiter well. A sample containing antigen is added to the well and allowed to react with
the antibody attached to the well, forming antigen-antibody complex. After the well is
washed, a second enzyme-linked antibody specific for a different epitope on the antigen is
added and allowed to react with the bound antigen. Then after unbound secondary antibody
is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme
to form colored products.
9.6.9 PROCEDURE OF SANDWICH ELISA
Prepare a surface to which a known quantity of antibody is bound.
Add the antigen-containing sample to the plate and incubate the plate at 37°c.
Wash the plate, so that unbound antigen is removed.
Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c.
Wash the plate, so that unbound enzyme-linked antibodies are removed.
Add substrate which is converted by the enzyme to produce a colored product.
Reaction of a substrate with the enzyme to produce a colored product.
9.6.10 ADVANTAGES
High specificity, since two antibodies are used the antigen is specifically captured and
detected.
Suitable for complex samples, since the antigen does not require purification prior to
measurement.
Flexibility and sensitivity, since both direct and indirect detection methods .
9.6.11 COMPETITIVE ELISA
This test is used to measure the concentration of an antigen in a sample. In this test, antibody
is first incubated in solution with a sample containing antigen. The antigen-antibody mixture
is then added to the microtitre well which is coated with antigen. The more the antigen
present in the sample, the less free antibody will be available to bind to the antigen-coated
well. After the well is washed, enzyme conjugated secondary antibody specific for isotype of
the primary antibody is added to determine the amount of primary antibody bound to the
well. The higher the concentration of antigen in the sample, the lower the absorbance.
9.6.12 PROCEDURE
Antibody is incubated with sample containing antigen.
Antigen-antibody complex are added to the microtitre well which are pre-coated with
the antigen.
Wash the plate to remove unbound antibody.
Enzyme linked secondary antibody which is specific to the primary antibody is added.
Wash the plate, so that unbound enzyme-linked antibodies are removed.
Add substrate which is converted by the enzyme into a fluorescent signal.
9.6.13 ADVANTAGES
High specificity, since two antibodies are used.
High sensitivity, since both direct and indirect detection methods can be used.
Suitable for complex samples, since the antigen does not require purification prior to
measurement.
VERY SHORT ANSWER TYPE QUESTIONS
Q1. Define widal test.
Q2. Write the full form of ASO.
Q3. Write the full form of CRP.
Q4. Define RF FACTOR.
Q5. Write the full form of VDRL.
Q6. Write the full form of ELISA.
Q7. . Write the full form of RF factor.
SHORT ANSWER TYPE QUESTIONS AND LONG ANSWER TYPE QUESTIONS
Q1.Discuss the principle and procedure of widal test
.Q2. Discuss the procedure and interpretation of widal test.
Q3. Discuss the principle and procedure of ASO test.
Q4. Discuss the procedure and interpretation of ASO test.
Q5 Discuss the principle and procedure of CRP test
Q6 Discuss the procedure and interpretation of CRP test.
Q7 Discuss the principle and procedure of RF test
Q8 Discuss the procedure and interpretation of RF test.
Q9 Discuss the principle and procedure of VDRL test
Q10 Discuss the procedure and interpretation of VDRL test.
Q11 Discuss the principle and procedure of ELISA test
Q12 Discuss the procedure and interpretation of ELISA test.
APPLICATION OF ELISA
1. Presence of antigen or the presence of antibody in a sample can be evaluated.
2. Determination of serum antibody concentrations in a virus test. 3. Used in food industry when detecting potential food allergens. 4. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird