Protocol for all basic laboratory tests: Biochemistry tests, microbiology tests, serology tests, histopathology tests

1. By,
B.VARSHINI
R.PRIYANKA
CLINICAL LABORATORY
TECHNIQUES

2. ELISA
1.Anti-Cardiolipin ELISA
It is an indirect solid phase immunoassay.
 PRINCIPLE:
 Highly purified cardiolipin is bound to microwells saturated with β2
glycoproteinI.
 Antibodies against these antigens, if present in diluted serum or plasma, bind to
respective antigen.
 Washing of microwells removes unspecific serum and plasma compounds.
Horseradish peroxidase (HRP) conjugated anti-human IgG and IgM
immunologically detect the bound patient antibodies forming conjugate Ag-Ab
complex.
 Washing of microwells removes unbound conjugate. An enzyme substrate in
presence of bound conjugate hydrolyses to form blue colour.
 The addition of an acid stops reaction forming yellow end product.
 The intensity of this yellow colour is measured photometrically at 450 nm.The
amount of colour is directly proportional to concentration of IgG and IgM
antibodies present in the original sample.

3.  MATERIALS REQUIRED:
 Microwell plate coated with anti-cardiolipin
 standard
 control
 Assay diluent
 Streptavidin- HRP
 Stop solution
 TMB substrate
 Microplate sealing film
 Wash concentrate(20 x)
 Micropipette and tips
 Preparation of sample buffer:
 Dilute the contents of each vial of the sample buffer concentrate(5x) with distilled
or deionised water to final volume 100ml prior to use. Store at 2-8°C in refrigerator
for atleast 30 days after preparation.

4.  Preparation of wash solution:
 Dilute the contents of each vial of the sample buffered wash solution concentrate (50x)
with distilled or deionised water to final volume of 1000ml prior to use. Store at 2-8°C
in refrigerator for atleast 30 days after preparation.
 Sample preparation:
 Dilute samples 1:100 with sample buffer before assay. Therefore combine 10µl of
sample with 990µl of sample buffer in polystyrene tube. Mix well and use.
 PROCEDURE:
 Prepare sufficient number of microplate modules to accommodate controls and
prediluted patient samples.
 Pipette 100µl of calibrators,controls, and prediluted patient samples in duplicate into
the wells. For determination of both IgG and IgM autoantibodies,calibrators,controls
and patient samples have to be pipetted in two attempts.
 Incubate for 30 min at room temp(20-28°C).
 Discard contents of microwells and wash 3 times with 300µl wash solution.
 Dispense 100µl of enzyme conjugate into each well.
 Incubate for 15 min at room temp.
 Discard contents of microwells and wash 3 times with 300µl of wash solution.
 Dispense 100µl of TMB substrate solution into each well.

5.  Incubate for 15 min at room temp.
 Add 100µl of stop solution to each well of modules and incubate for 15 min at room
temp.
 Read absorbance on ELISA Reader at 450 nm within 10 min of adding stop solution.
 CLINICAL SIGNIFICANCE:
Increased cardiolipin leads to Systemic Lupus Erythematosus,
Thrombocytopenia,chorea, Recurrent abortion, Thrombosis, Cerebral ischemia,
Epilepsy and Intrauterine death.
 INTERPRETATION:
IgG ,IgM
Negative Value: <10
Positive Value: >10

6. 2.ANTI-PHOSPHOLIPID ELISA
 Mixture of highly purified cardiolipin, phosphatidyl serine, phosphatidyl inositol,
phosphatidic acid & human β2 glycoprotein I is bound to microwells.
PROCEDURE:
 Prepare sufficient number of microplate modules to accommodate controls and
prediluted patient samples.
 Pipette 100µl of calibrators,controls and prediluted patient samples in duplicate into the
wells. For determination of both IgG and IgM autoantibodies,calibrators,controls and
patient samples have to be pipetted in two attempts.
 Incubate for 30 min at room temp(20-28°C).
 Discard contents of microwells and wash 3 times with 300µl wash solution.
 Dispense 100µl of enzyme conjugate into each well.
 Incubate for 15 min at room temp.
 Discard contents of microwells and wash 3 times with 300µl of wash solution.

7.  Dispense 100µl of TMB substrate solution into each well.
 Incubate for 15 min at room temp.
 Add 100µl of stop solution to each well of modules and incubate for 15 min at room
temp.
 Read absorbance on ELISA Reader at 540 nm within 10 min of adding stop solution.
INTERPRETATION:
IgG ,IgM
Negative Value: <7
Positive Value: >7
CLINICAL SIGNIFICANCE:
Increased phospholipid leads to Systemic Lupus Erythematosus.

8. 25(OH) VITAMIN D ELISA
 Vitamin D is a steroid hormone involved in the active intestinal absorption of
calcium and in the regulation of its homeostasis.
 Vitamin D has two isomers : vitamin D2 & Vitamin D3. Vitamin D2 is obtained
from dairy products whereas Vitamin D3 is produced in the skin after exposure
to ultraviolet light.
 In the liver, Vitamin D is hydroxylated at its carbon 25 to form 25-OH Vitamin
D. This metabolite is the predominant circulating form of Vitamin D & is
considered to be an accurate indicator of the general Vitamin D status of an
individual.
 PRINCIPLE:
 It is a solid phase ELISA, based on the principle of competitive binding.
 Anti-Vitamin D antibody coated wells are incubated with Vitamin D standards,
controls, samples & Vitamin D-Biotin conjugate at room temp for 90min.
 During the incubation, a fixed amount of biotin-labelled Vitamin D competes
with endogenous Vitamin D in the sample, standard or quality control serum
for a fixed number of binding sites on the anti-Vitamin D antibody. Following a
wash step, bound vitamin D-Biotin is detected with streptavidin-HRP (SA-
HRP). SA-HRP conjugate immunologically bound to the well progressively
decreases as a concentration of vitamin D in the specimen increases.

9.  Unbound SA-HRP conjugate is then removed and the wells are washed. Next, a
solution of TMB reagent is added and incubated at room temp for 30min,
resulting in the development of blue colour.
 The colour development is stopped with addition of stop solution, and the
absorbance is measured spectrophotometrically at 450nm.
 The colour intensity will be inversely proportional to the amount of 25-OH
Vitamin D in the sample. The assay measures both the 25-OH Vitamin D2 &
Vitamin D3. The total assay procedure run time is 2.5h.
 INTERPRETATION:
Deficient : <10 ng/ml
Insufficient : 10-30 ng/ml
Sufficient : 30-100ng/ml
Intoxication : > 100ng/m
 CLINICAL SIGNIFICANCE:
 Vitamin D deficiency leads to osteoporosis, Rickets, Osteomalacia, Cancer and
Cardiovascular diseases. Thus quantification of vitamin D in serum/plasma is
used as marker.

10. ANTI-MULLERIN HORMONE(AMH) ELISA
 AMH is a member of TGFβ Superfamily, homodimeric glycoprotein composed of two
55kDa N-terminal & two 12.5kDa C-terminal homodimers, non-covalently linked by
disulphide bridges. The cleaved AMH non-covalent complex binds to AMH RII &
stimulates intracellular signalling whereas full length AMH shows only minimal
activity.
 PRINCIPLE:
 AMH ELISA is quantitative sandwich type.
 In this assay, calibrators, controls and samples are incubated in microtitration wells
which have been coated with anti-AMH antibody.
 After incubation and washing,anti-AMH detection antibody labeled with biotin is
added to each well. After a second incubation and washing step, streptavidin-
horseradish peroxidase (HRP)is added to the wells. After a third incubation and
washing step, the substrate tetramethylbenzidine (TMB) is added to the wells.
 Lastly, an acidic stopping solution is added. The degree of enzymatic turnover of the
substrate is determined by dual wavelength absorbance measurement at 450 nm and
between 600 and 630nm.
 The absorbance measured is directly proportional to the concentration of AMH in the
samples.

11.  INTERPRETATION:
MALES:
0-13 days : 15.5 - 48.7
14 days to 11 months : 39.1-91.1
1yr to 6yrs :48.0 - 83.2
7-8yrs : 33.8 – 60.2
Adults : 0.6 – 13.7
FEMALES:
0-8Yrs : 0.0 – 0.71
Adults : 0.19 – 9.13
 CLINICAL SIGNIFICANCE:
AMH is secreted by sertoli cells in males. During embryonic development, AMH is
responsible for Mullerian Duct regression. AMH continues to be produced by the
testis until puberty and decreases slowly to residual post-puberty values. In females,
AMH is produced by the granulosa cells of small growing follicles from 36th week of
gestation onwards until menopause when levels become undetectable. Low end anti-
Mullerian hormone leads to premature ovarian insufficiency, ovarian tumor&
menopause.

12. HIV ELISA:
 PRINCIPLE:
 Microlisa HIV (Ag &Ab) test is based on sandwich type ELISA. HIV
envelope proteins gp41, C-terminus of gp120 for HIV-1 & gp36 for HIV-2
representing immunodominant epitopes & anti-HIV1 p24 Abs are coated onto
microtiter wells. Specimens & controls are added to wells & incubated.
 Antibodies to HIV-1 & HIV-2 if present in specimen, will bind to specific antigens
absorbed onto the surface of the wells. The plate is then washed to remove unbound.
HRP conjugate gp41, C-Terminus of 120 HIV1 & gp36 of HIV2 & anti-HIV1 p24
antibodies is added to each well.
 The conjugate will bind to HIV Ag-Ab or anti p24 Ab-Ag complex. Substrate,
chromogen& hydrogen peroxide are added & incubated. Then absorbance is
measured spectrophotometrically at 450nm.
REAGENT PREPARATION:
 Wash Solution:
100ml of wash solution is prepared by adding 96ml of Distilled water with 4ml of
wash buffer.

13.  Substrate Preparation:
200µl of TMB substrate was added to 200µl of substrate diluent & mixed well.
 Conjugate Preparation:
1ml of conjugate diluent was mixed with 10µl of conjugate activator(enzyme
conjugate concentrate).
 PROCEDURE:
1. Dispense 25µl of sample diluent into each well.
2. Dispense 100µl of samples and controls into each well.
3. Cover the plate and incubate the wells at 37°C for 1 hour.
4. Dispense 300µl of wash buffer into each well for 6 times.
5. Dispense 100 µl of conjugate into each well.
6. Incubate at room temp for 30 min .
7. Dispense 300µl of wash buffer into each well and repeat it for 6 times.
8. Dispense 100µl of TMB substrate into each well.

14. 9. Incubate at room temp for 30 min in dark.
10.Dispense 100µl of stop solution into each well to stop the enzymatic reaction.
11.Read absorbance on ELISA Reader at 450 nm within 10 min of adding stop solution
.
 INTERPRETATION:
Negative : <0.16
Positive : >0.16

15. HISTOPATHOLOGY
Histopathology refers to the microscopic examination of tissue in order to study the
manifestations of disease. In clinical medicine, histopathology refers to the
examination of a biopsy or surgical specimen by a pathologist, after the specimen
has been processed and histological sections have been placed onto glass slides.
Steps involved are :
 Fixation
 Dehydration
 Clearing
 Embedding
 Microtomy(sectioning)
 Staining
 Preparation of permanent mounts

16.  FIXATION
The purpose of fixation is to preserve tissues permanently in as life-like a
state as possible. Fixation should be carried out as soon as possible after
removal of the tissues (in the case of surgical pathology) or soon after death
(with autopsy) to prevent autolysis. There is no perfect fixative, though
formaldehyde comes the closest. Therefore, a variety of fixatives are
available for use, depending on the type of tissue present and features to be
demonstrated.
 FIXATION - FACTORS AFFECTING FIXATION
 Buffering – neutral pH (6-8)
 Penetration –Formalin and alcohol penetrates best(sections should be 2-3mm
thin)
 Volume - 10:1 ratio of fixative to tissue
 Temperature- Increasing the temperature will increase the speed of reaction
 Concentration- Formalin is best at 10%.
 Time interval- if time exceeds, cellular organelles will be lost, nuclear
shrinkage and artefactual clumping will occur.

17. • DEHYDRATION
Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with
paraffin. First, the water from the tissues must be removed by dehydration.
This is usually done with a series of alcohols, say 70% to 95% to 100%.
Sometimes the first step is a mixture of formalin and alcohol.
• CLEARING
 During dehydration water in tissue has been replaced with alcohol.
 The next step alcohol should be replaced by paraffin wax.
 As paraffin wax is not alcohol soluble, we replace alcohol with a substance
in which wax is soluble. This step is known as clearing. Xylene is most
commonly used clearing agent.

18. • EMBEDDING
 Paraffin wax is routinely used .
 Tissues are placed in moulds with their labels and fresh melted wax is poured in it
and allowed to settle and solidify.
 Once the block has cooled sufficiently , it should be immersed in cold water to cool
it rapidly.
 After the block has completely cooled it is cut into individual blocks and each is
trimmed.
 Labels are made to adhere on the surface of the block by melting the wax with a
metal strips sufficiently warmed.

19.  SECTIONING
 Once the tissues have been embedded, they must be cut into sections that can be
placed on a slide. This is done with a microtome. The microtome is nothing more than
a knife with a mechanism for advancing a paraffin block standard distances across it.
For proper sectioning very sharp knife is necessary.
 Knives are either of the standard thick metal variety or thin disposable variety. The
former type is expensive than the latter. The advantage of the disposable blade
becomes apparent when sectioning a block in which is hidden a metal wire or suture.
 Microtomes have a mechanism for advancing the block across the knife. Usually this
distance can be set, for most paraffin embedded tissues at 6 to 8 microns

20.  Once sections are cut, they are floated on a warm water bath that helps remove
wrinkles.
 Then they are picked up on a glass microscopic slide. The glass slides are then
placed in a warm oven for about 15 minutes to help the section adhere to the
slide.
 STAINING
 The embedding process must be reversed in order to get the paraffin wax out of
the tissue and allow water soluble dyes to penetrate the sections. Before
staining, the slides are "deparaffinized" by running them through xylenes (or
substitutes) to alcohols to water. There are no stains that can be done on tissues
containing paraffin.
 The staining process makes use of a variety of dyes that have been chosen for
their ability to stain various cellular components of tissue. The routine stain is
that of hematoxylin and eosin (H and E).

21.  Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the
tissues.
 Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell
nucleus.
 Hematoxylin stains are either "regressive" or "progressive". With a regressive stain,
the slides are left in the solution for a set period of time and then taken back through a
solution such as acid-alcohol that removes part of the stain. This method works best
for large batches of slides to be stained and is more predictable on a day to day basis.
 With a progressive stain the slide is dipped in the hematoxylin until the desired
intensity of staining is achieved, such as with a frozen section.
 Eosin is an acidic dye with an affinity for cytoplasmic components of the cell.

22.  MOUNTING
 Few drops of DPX mount is placed on the stained section of the slide and is
covered with a thin piece plastic or glass to protect the tissue from being scratched,
to provide better optical quality for viewing under the microscope, and to preserve
the tissue section for years to come.
 Bubbles under the coverslip may form when the mounting media is too thin.
 Contamination of clearing agents or coverslipping media may also produce a
bubbled appearance under the microscope.

23. LIQUID BASED CYTOLOGY TEST
This procedure demonstrates atypical, inflammatory & malignant cells in
cytology smears.
PROCEDURE
 Sample preparation:
2ml cleaning solution is added to 5 ml sample (cervix liquid) and
centrifuged for 20 mins. Supernatant is discarded and few drops of
cell fixate is added to the pellet & mixed well.
 PAP Smear preparation:
100µl of sample is taken and smeared in slide and dried.
 PAP Staining:
 The slide is dipped in 70% alcohol for 5 min
 Then the slide is dipped in 50% alcohol for 1 min.
 The slide is then rinsed in tap water
 Then it is rinsed in distilled water for 1 min
 Then slide is dipped in hematoxylin stain for 3-5 min
 This is again washed in tap water for 3min

24.  The slide is then dipped in alcohol 1 for 20sec
 Then slide is dipped in OG-6 cytology stain for 2 min.
 This is again dipped in 95% alcohol 2 for 20 sec
 EA 50 polychromatic solution for 3 min
 The slide has to meet 3 changes of alcohol solution 1, 2, 3 in which it has to be
dipped in each for 20 sec
 The slide is dried
 The slide is dipped in xylene for 20 min and dried
 Few drops of DPX mount is placed on the stained section of the slide and is
covered with coverslip.
 Then the slide is observed under microscope.
• Interpretation:
 Chromatin-blue
 Keratin-orange
 Superficial squamous cells-various shades of pink
 Nucleoli, cilia, RBCs- various shades of pink
 All metabolic cell cytoplasm- various shades of blue-green

25. MICROBIOLOGY
 ACID FAST STAINING
 It is the differential staining techniques which was first developed by Ziehl and later
on modified by Neelsen.
 The main aim of this staining is to differentiate bacteria into acid fast group and non-
acid fast groups.
 This method is used for those microorganisms which are not staining by simple or
Gram staining method, particularly the member of genus Mycobacterium, are
resistant and can only be visualized by acid-fast staining.
 PRINCIPLE
 When the smear is stained with carbolfuchsin, it solubilizes the lipoidal material
present in the Mycobacterial cell wall but by the application of heat, carbolfuchsin
further penetrates through lipoidal wall and enters into cytoplasm.
 Then after all cell appears red. Then the smear is decolorized with decolorizing agent
(3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of
large amount of lipoidal material in their cell wall which prevents the penetration of
decolorizing solution.

26.  The non-acid fast organism lack the lipoidal material in their cell wall due to which
they are easily decolorized, leaving the cells colorless.
 Then the smear is stained with counterstain, methylene blue. Only decolorized cells
absorb the counter stain and take its color and appear blue while acid-fast cells
retain the red color.
PROCEDURE

28. BIOCHEMICAL TESTS:
TRIPLE SUGAR IRON AGAR (TSI)
 PRINCIPLE:
Triple sugar iron agar test is used to determine whether gram negative bacilli utilise
glucose and lactose or sucrose fermentatively and produce hydrogen sulfide (H2S).
 Composition of Triple Sugar Iron Agar (TSI)
Lactose, Sucrose and Glucose in the concentration of 10:10:1 (i.e. 10 part Lactose,
10 part Sucrose and 1 part Glucose).
Iron: Ferrous sulfate: Indicator of H2S formation
Phenol red: Indicator of acidification (It is yellow in acidic condition and red under
alkaline conditions).
It also contains Peptone which acts as source of nitrogen (whenever peptone is
utilized under aerobic condition ammonia is produced).
TSI Tube contains contains butt (poorly oxygenated area on the bottom) slant (angled
well oxygenated area on the top).
 PROCEDURE:
 With a sterilized straight inoculation needle touch the top of a well-isolated colony
 Inoculate TSI Agar by first stabbing through the center of the medium to the bottom
of the tube and then streaking the surface of the agar slant.
 Leave the cap on loosely and incubate the tube at 35 in ambient air for 18 to 24
hours.

29.  INTERPRETATION:
 If lactose (or sucrose) is fermented, a large amount of acid is produced, which
turns the phenol red indicator both in butt and in the slant. Some organisms
generate gases, which produces bubbles/cracks on the medium.
 If lactose is not fermented but the small amount of glucose is, the oxygen
deficient butt will be yellow (remember that butt comparatively have more
glucose compared to slant i.e. more media more glucose), but on the slant the
acid (less acid as media in slant is very less) will be oxidized to carbon dioxide
and water by the organism and the slant will be red (alkaline or Neutral pH).
 If neither lactose/sucrose nor glucose is fermented, both the butt and the slant
will be red. The slant can become a deeper red-purple (more alkaline) as a result
of production of ammonia from the oxidative deamination of amino acids
(remember peptone is a major constituents of TSI Agar) .
 If H2S is produced, the black colour of ferrous sulfide is seen.

30. ORGANISM TRIPLE SUGAR IRON AGAR
BUTT SLANT GAS H2S
E.coli A A + -
Shigella spp A K - -
RESULT

31. INDOLE TEST:
 PRINCIPLE:
 Indole test is used to determine the ability of an organism to spilt amino acid
tryptophan to form the compound indole.
 Tryptophan is hydrolysed by tryptophanase to produce three possible end products –
one of which is indole.Indole production is detected by Kovac’s or Ehrlich’s reagent
which contains 4 (p)-dimethylaminobenzaldehyde, this reacts with indole to produce
a red coloured compound.
 Indole test helps to differentiate Enterobacteriaceae and other genera.
 Two methods are in used:
 a spot indole test, which detects rapid indole producing organisms and
 a conventional tube method requiring overnight incubation, which identifies weak
indole producing organisms.
 PROCEDURE:
a. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the
test organism in tryptophan broth.
b. Incubate at 37°C for 24-28 hours in ambient air.
c. Add 0.5 ml of Kovac’s reagent to the broth culture.

32. RESULT:
NEGATIVE -Klebsiella pneumonia
POSITIVE - Klebsiella oxytoca

33. METHYL RED TEST (MR)
 PRINCIPLE:
 Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation
when supplied glucose. Types and proportion of fermentation products produced by anaerobic
fermentation of glucose is one of the key taxonomic characteristics which help to differentiate
various genera of enteric bacteria.
 Mixed acid fermentation is one of the two broad patterns, 2-3-butanediol fermentation being
another. In mixed acid fermentation, three acids (acetic, lactic and succinic) are formed in
significant amounts. The mixed acid pathway gives 4 mol of acidic products (mainly lactic and
acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol of
H2 per mol of glucose fermented.
 These large amounts of acid results significant decrease in the pH of the medium below 4.4.
This is visualized by using pH indicator, methyl red (p-dimethylaminoaeobenzene-O-
carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4.
 The pH at which methyl red detects acid is considerably lower than the pH for other indicators
used in bacteriologic culture media. Thus, to produce a colour change, the test organism must
produce large quantities of acid from carbohydrate substrate being used.
 Procedure for Methyl Red (MR) Test
 MR-VP broth is used for both MR Test and VP test. Only the addition of reagent differs, and
both tests are carried out consecutively.
 Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under
investigation.

34.  INTERPRETATION:
 MR Positive: When the culture medium turns red after addition of methyl red,
because of a pH at or below 4.4 from the fermentation of glucose.
 MR Negative: When the culture medium remains yellow, which occurs when less
acid is
 produced (pH is higher) from the fermentation of glucose
RESULT:
POSITIVE – Salmonella typhii
NEGATIVE – Vibrio cholera
VARIABLE – Staphylococcus aureus

35. VOGES-PROSKAUER (VP) METHOD
 PRINCIPLE:
 Voges-Proskauer is a double eponym, named after two microbiologists working at
the beginning of the 20th century. They first observed the red colour reaction
produced by appropriate culture media after treatment with potassium hydroxide. It
was later discovered that the active product in the medium formed by bacterial
metabolism is acetyl methyl carbinol, a product of the butylenes glycol pathway.
 Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is
further metabolized through various metabolic pathways, depending on the enzyme
systems possessed by different bacteria. One such pathways result in the production
of acetion (acetyl methyl carbinol), a neutral-reacting end product.
 Organisms such as members of the Klebsiella-Enterobacter-Hafnia-Serratia group
produce acetoin as the chief end product of glucose metabolism and form smaller
quantities of mixed acids. In the presence of atmospheric oxygen and 40% potassium
hydroxide, acetoin is converted to diacetyl, and alpha-naphthol serves as a catalyst to
bring out a red complex.

36.  PROCEDURE :
 Inoculate a tube of MR/VP broth with a pure culture of the test organism.
 Incubate for 24 hours at 35oC
 At the end of this time, aliquot 1 mL of broth to clean test tube.
 Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH. (Note: It is
essential that the reagents be added in this order.)
 Shake the tube gently to expose the medium to atmospheric oxygen and allow the
tube to remain undisturbed for 10 to 15 minutes.

37.  INTERPRETATION:
 VP Positive: Thisis represented by the development of a red color 15 minutes or
more after the addition of the reagents indicating the presence of diacetyl, the
oxidation product of acetoin .
 VP Negative: Yellow or copper color at the surface of the medium (acetoin absent)
 RESULT:
POSITIVE – Serratia marcescens
NEGATIVE – Proteus vulgaris
VARIABLE – Enterococcus faecalis

38. UREA HYDROLYSIS/ UREASE TEST (CHRISTENSEN’S METHOD)
 PRINCIPLE:
 Urea hydrolysis/Urease test is used to detect the ability of an organism to produce
urease that hydrolyses urea contained in the medium. Hydrolysis of urea produces
ammonia and carbon dioxide The formation of ammonia makes the medium alkaline
and the shift in pH is detected by the change of color of indicator phenol red from
light orange (pH 6.8) to pink or magenta color (pH 8.1).
 PROCEDURE:
 Pick the organism from an isolated colony and streak the surface of urea agar or
inoculate slant with 1 or 2 drops of overnight incubated broth.
 Leave the cap loose and incubate at 35oC for 48 hours to 7 days.

39.  INTERPRETATION:
 Positive: change in color of slant from light orange to pink or magenta
 Negative: No change in color (color of slant and butt remains light orange)
 RESULT:
POSITIVE – Proteus mirabilis
NEGATIVE – Salmonella paratyphii A
Salmonella paratyphii B
VARIABLE _ Citrobacterkoseri

40. CITRATE UTILISATION TEST
 PRINCIPLE:
 Citrate utilisation test is used to detect the ability of an organism to utilize sodium
citratre as a sole source of carbon and ammonium salt as a sole source of nitrogen.
Bacteria that grow in the medium turn the medium alkaline. This is indicated by the
change of color of bromothymol blue indicator from green to blue.
PROCEDURE:
 Inoculate Simmons citrate agar on the slant by touching a colony that is 18-24 hrs
old with a straight wire.There is no need to stab the butt of the medium. Do
not inoculate from the broth culture because the inoculum will be too heavy.
 Incubate at 35oC-37oC for up to 7 days.
 Observe the blue coloration of the media.

41. INTERPRETATION:
Positive: Growth on the medium, with or without the change in color of the medium
(blue).
Negative: Absence of growth
RESULT:
POSITIVE – Pseudomonas aeruginosa
NEGATIVE – Staphylococcus aureus
VARIABLE –Proteus vulgaris

42. BILE-ESCULIN TEST
 Bile-esculin test is widely used to differentiate enterococci and group D
streptococci, which are bile tolerant and can hydrolyze esculin to esculetin, from
non-group D viridans group streptococci, which grow poorly on bile. It is a low
cost, rapid test with good sensitivity and specificity (>90%).
 PRINCIPLE:
 Bile-esculin test is based on the ability of certain bacteria, notably the group D
streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile
(4% bile salts or 40% bile).
 Note: Many bacteria can hydrolyze esculin, but few can do so in the presence of
bile.
 Esculin is a glycosidiccoumarin derivative (6-beta-glucoside-7-hydroxy-coumarin).
 The two moieties of the molecule (glucose and 7-hydroxycoumarin) are linked
together by an ester bond through oxygen. For this test, esculin is incorporated into a
medium containing 4% bile salts.
 Bacteria that are bile-esculin positive are, first of all, able to grow in the presence of
bile salts. Hydrolysis of the esculin in the medium results in the formation of
glucose and a compound called esculetin.

43.  Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium
component ferric citrate) to form a black diffusible complex.
 Group D streptococci and enterococci include opportunistic pathogens such
as Enterococcus faecalis, Enterococcus faecium, and Streptococcus bovis.
 COMPOSITION:
 Bile-esculin agar medium is prepared as agar slants or plates.
 The constituent of Bile-esculin agar medium are: Peptone, Beef extract, Oxgall (Bile),
Esculin, Ferric citrate and Agar. Bile esculin medium contains esculin and peptone for
nutrition and bile to inhibit Gram-positive bacteria other than Group D streptococci
and enterococci. Ferric citrate is added as a color indicator.
 PROCEDURE:
 With an inoculating wire or loop, touch two or three morphologically similar
streptococcal colonies and inoculate the slant of the bile esculin medium with an S-
shaped motion, or streak the surface of a bile esculin plate for isolation. (Note: There
is no need to stab the medium.)
 The inoculated tube is incubated at 35-37 degree Celsius for 24 hours and the results
are determined.

44.  INTERPRETATION:
Diffuse blackening of more than half of the slant within 24-48
hours indicatesesculin hydrolysis. On plates, black haloes will be observed around
isolated colonies and any blackening is considered positive. All group D
streptococci will be bile-esculin positive within 48 hours.

45. COAGULASE TEST :
 Coagulase test is done to distinguish Staphylococcus aureus from other Coagulase
negative Staphylococci (CONS) like Staphylococcus epidermidis, Staphylococcus
saprophyticus etc.
 Staphylococcus aureus produces two types of coagulase, i.e. free coagulase and
bound coagulase. Free coagulase is an extracellular enzyme that can be detected in
tube coagulase test while bound coagulase is a cell wall associated protein that can
be detected in slide coagulase test.
SLIDE COAGULASE TEST
PRINCIPLE:
Bound coagulase is also known as clumping factor. It cross-links α and β chain of
fibrinogen in plasma to form fibrin clot that gets deposited on the cell wall of the
cocci. As a result, individual coccus sticks to each other and clumping can be
observed.

46.  PROCEDURE:
 Emulsify few colonies of Staphylococci from culture in a drop of normal saline on
two ends of clean glass slide.
 Label one as “test” and the other as “control”. The control suspension serves to
rule out false positives due to autoagglutination.
 Mix a drop of rabbit or human plasma with the test suspension.
 Observe agglutination or clumping of cocci.
 INTERPRETATION:
Agglutination within 5-10 seconds is considered as positive. Some strains
of S.aureus may not produce bound coagulase, and such strains must be identified by
tube coagulase test.

47. TUBE COAGULASE TEST
PRINCIPLE:
The free coagulase secreted by S. aureus reacts with coagulase reacting
factor (CRF) present in plasma to form a complex, thrombin. This converts
fibrinogen to fibrin resulting in clotting of plasma.
PROCEDURE:
 Take three test tubes and label them as “test”, “negative control” and
“positive control”.
 Fill each test tube with 1 ml of 1:6 dilution of human plasma in normal
saline.
 Add 0.1 ml of overnight broth culture to the tube labeled test. Also add 0.1
ml of overnight broth culture of known S. aureus to the tube labeled
positive control and 0.1 ml of sterile broth to the tube labeled negative
control.
 Incubate all the tubes at 37oC and observe up to four hours.

48. INTERPRETATION:
Positive result is indicated by clotting of the plasma, which remains in place even after
inverting the tube. If the test remains negative until four hours at 37oC, leave the tube
at room temperature for overnight incubation. Some strains on continued incubation
produce fibrinolysin that lyses the clot.
Slide coagulase test is useful as a screening test while tube coagulase test is useful in
confirmation of coagulase test .

49. OXIDASE TEST:
PRINCIPLE:
The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an
enzyme of the bacterial electron transport chain.
When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-
phenylenediamine) to (indophenols)purplecolor end product. When the enzyme is not
present, the reagent remains reduced and is colorless.
PROCEDURE:
1.Oxidase reaction is carried out by touching and spreading a well isolated colony
on the oxidase disc.
2.The reaction is observed within 5-10 seconds at 25-30°C.
3.A change later than 10 seconds or no change at all is considered negative reaction.
INTERPRETATION:
POSITIVE: Deep purplish blue colouration of disc
NEGATIVE: No colour change
RESULT:
POSITIVE – Vibrio cholerae
NEGATIVE – E.coli

50. CATALASE TEST :
PRINCIPLE:
Catalase test is done to check for the presence of enzyme catalase in bacteria that
hydrolyzes hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). If the
bacteria possess catalase enzyme, it’s evident by formation of bubbles in the test due
to liberation of oxygen. Lack of catalase is indicated by absence of bubble
production.
PURPOSE:
 It is used to differentiate between genus Staphylococcus and Streptococcus.
Staphylococcus possesses catalase (catalase positive) while Streptococcus lacks this
enzyme (catalase negative).
 It is used for presumptive identification of gram negative bacilli belonging to
Enterobacteriaceae family.
PROCEDURE:
 Using a sterile loop or wooden stick, pick a small amount colony and place it onto
the clean, dry glass slide.
 Using a dropper, place a drop of 3% hydrogen peroxide onto the organism on the
microscope slide.
 Observe for the formation of bubbles.

51. INTERPRETATION:
Positive: Copious bubbles produced
Negative: No or few bubbles produced
Note: Enterococci produces an enzyme peroxidase that slowly catalyses the breakdown of
H2O2 and the test may appear weakly positive. This reaction is not a truly positive test.
RESULT:
POSITIVE – Providencia spp
NEGATIVE – Acinetobacter spp

52. MANNITOL MOTILITY TEST:
Mannitol Motility Test Medium is a semisolid medium suitable for determining motility
and mannitol fermentation.
PRINCIPLE:
Mannitol Motility Test Medium is designed to differentiate bacteria on the basis of their
motility and ability to ferment mannitol (1). The highly nutritious peptic digest of
animal tissue supports luxuriant growth of fastidious bacteria like Staphylococci.
COMPOSITION:
Chemicals Gms / Litre
Peptic digest of animal tissue 20.000
Mannitol 2.000
Potassium nitrate 1.000
Phenol red 0.040
Agar 3.000
Final pH ( at 25°C) is 7.6±0.2

53. PREPARATION OF MEDIA:
Suspend 26.04 grams in 1000 ml distilled water. Heat to boiling to dissolve the
medium completely. Dispense into test tubes. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes. Cool the tubed medium in an upright position.
PROCEDURE:
1.Inoculate tubes with a pure culture by stabbing the center of the column of medium to
greater than half the depth.
2. Incubate tubes for 24-48 hours at 35 ± 2°C in an aerobic atmosphere.
INTERPRETATION:
Semisolid nature of the medium due to 0.3%
agar helps to detect motility. Motile bacteria
produce diffused growth throughout the
medium while non-motile bacteria grow only
along the line of inoculation. Fermentation of
mannitol produces acidity in the medium.
Phenol red is the pH indicator, which detects
acidity by exhibiting a visible colour change
from red to yellow

54. BLOOD CULTURE
 A blood culture is a test to find an infection in the blood. The blood does not
normally have any bacteria or fungi in it. A blood culture can show what
bacteria or fungi are in the blood.
 A bacterial infection in the blood, called bacteremia, can be serious because
the blood can spread the bacteria to any part of the body. A blood infection
most often occurs with other serious infections, such as those affecting the
lungs, kidneys, bowel, gallbladder , or heart valves.
 A blood infection may also develop when the immune system is weak. This
can occur in infants and older adults, and from disease (such as cancer or
AIDS) or from medicines (such as corticosteroids or chemotherapy) that
change how well your body can fight infections (immunity).

55.  PURPOSE:
 A blood culture is done to:
 Find a bacterial infection that has spread into the blood, such as
meningitis, osteomyelitis, pneumonia, a kidney infection, or sepsis. A culture can also
show what type of bacteria is causing the infection.
 Find a fungal infection, such as yeast, in the blood.
 Check for endocarditis, which is an infection of the valves of the heart .
 Find the best antibiotics to kill the bacteria or fungi. This is called sensitivity testing.
 Find the cause of an unexplained fever or shock or a person becoming extremely ill.

56. AUTOMATED BLOOD CULTURE (BACTEC™ 9050)
The BACTEC 9000 series of blood culture instruments are designed for the rapid detection of
microorganisms in clinical specimens. The sample to be tested is inoculated into the vial which
is entered into the BACTEC instrument for incubation and periodic reading. Each vial contains
a sensor which responds to the concentration of CO2 produced by the metabolism of
microorganisms or the consumption of oxygen needed for the growth of microorgnisms. The
sensor is monitored by the instrument every ten minutes for an increase in its fluorescence,
which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present
in the vial. A positive reading indicates the presumptive presence of viable microorganisms in
the vial.

57. RESULTS:
A blood culture is a test to find an infection in the blood. Most bacteria can be seen in the
culture in 2 to 3 days, but some types can take 10 days or longer to show up. Fungus can
take up to 30 days to show up in the culture.
If bacteria are found in the culture, another test is often done to find the best antibiotic that
will kill the bacteria. This is called sensitivity or susceptibility testing. Sensitivity testing is
important so the blood infection is treated correctly. This also helps prevent bacteria from
becoming resistant to antibiotics
Normal: No bacteria or fungus is found. Normal culture results are called negative.
Abnormal: Bacteria or fungus grows in the culture. Abnormal culture results are called positive.
FACTORS AFFECTING THE TEST:
 If you have taken antibiotics recently. These medicines may stop the growth of bacteria in
the culture.
 If the blood sample is contaminated by bacteria or fungus on the skin.
 If the blood test misses the time when bacteria actually are in the blood. Blood culture tests
are done at several different times to make sure bacteria are not missed.
 If the blood test is not done correctly or the blood sample is not processed properly. In these
cases, a false-positive or false-negative result could occur.

58. NOTE:
 Some types of bacteria infect the blood when another infection of the kidneys, throat,
lungs, or another part of the body is present. This may not mean a serious infection of
the blood.
 About 5% of blood cultures are contaminated with normal skin bacteria (a type of
staph bacteria). So it is sometimes hard to see whether the bacteria that grow in the
culture are the cause of the blood infection or not. This is why more than one blood
sample is taken. When the same bacteria grow in several blood cultures, it is likely
that those bacteria are in the blood and are causing the infection. When staph bacteria
grow in the culture in less than 48 hours, it is likely that the staph bacteria are in the
blood and are causing the infection.
 A culture that does not grow any bacteria does not always mean a blood infection is
not present. The amount of blood taken, the timing of the blood sample, the type of
culture done, and recent use of antibiotics can affect the growth of bacteria in the
culture.