Laboratory diagnosis of Bacterial Infections: Direct Detection

Direct detection of pathogen in the clinical specimen plays a very important role in early institution
of antimicrobial therapy. These include microscopic demonstration of pathogens in the clinical
specimens and other methods including detection of antigen or nucleic acid in specimen.
A) Microscopic Examination of clinical specimen:
The reasons for performing microscopic examination of clinical materials are as below.
i) The number and percentage of segmented neutrophils that are present indicate the
magnitude and type of inflammatory response. The quality of specimen can also be
validated.
ii) The observation of bacteria, hyphae and/or yeast forms, parasitic structures or viral
inclusions may provide sufficient information to render an immediate presumptive
diagnosis.

Microscopic examination can be done in two ways-

a) Examination of stained preparation
b) Examination of unstained preparation

1. Examination of Stained preparation:

Structural details of cells and organisms cannot be appreciated under a light microscope due to
lack of contrast. So, it is necessary to enhance contrast for better visibility of cells and organisms.
Various staining techniques used for direct demonstration of pathogens are as follows-
i) Gram stain
ii) Albert stain
iii) Acid fast stain
iv) Negative stain
v) Impregnation stain
vi) Bipolar stain
vii) Simple stain
viii) Fluorescent stain

I) Gram stain:
It is most commonly employed staining technique in diagnostic microbiology. It is a differential
stain used frequently to differentiate between gram positive (purple) and gram negative (reddish
pink) organisms. It may also be used to determine whether a specimen is representative of the site
of infection. It may be evaluated by watching segmented neutrophil count in the specimen.
This technique has been applied to the evaluation of sputum samples by two well-known grading
systems-

 Bartlett’s grading systems:

Bartlett has devised a grading system for evaluating sputum samples for the relative number of
squamous epithelial cells and segmented neutrophils in direct gram stained sputum samples.
Number of Neutrophils per low power field Grade
(10X)
<10 0
10-25 +1
>25 +2
Presence of mucous +1
Number of Epithelial cells per low power Grade
field (10X)
10-25 -1
>25 -2
Total

Average for the number of epithelial cells and neutrophils in about 20-30 separate 10X
microscopic fields is to be determined. Then a total is to be calculated.
A final score of ‘0’ or less indicates lack of active inflammation or contamination with saliva.
Unsatisfactory specimens should be rejected and a properly collected repeat sputum specimen is
to be requested.

 Murray and Washington’s Grading system:

A similar grading system has been proposed by Murray and Washington. The large number of
epithelial cells in groups 1 to 4 of this system indicates contamination with oropharyngeal
secretions and invalidates the samples. Only group 5 specimens are considered clinically relevant.
Epithelial cells per low Neutrophils per low power
power field (10X) field (10X)
Grade 1 25 10
Grade 2 25 10-25
Grade 3 25 25
Grade 4 10-25 25
Grade 5 <10 25
In case of lower respiratory tract infection with endotracheal intubation or lower urinary tract
infection with Foley’s catheter in-situ or repeated intermitted catheterization, mere presence of
leukocytes cannot be used as an indication of a clinically important infection.

Gram stained smear showing squamous epithelial cells in sputum under low power field (10 X)

Gram stained smear showing segmented neutrophils in sputum under low power field (10 X)
 II) Albert Stain:
It is used to demonstrate the metachromatic granules of Corynaebacterium diphtheriae. It is a
type of staining in which staining reagents used stain the constituents of the cells and tissues
in various shades of their own fundamental colour. This colour shift is called metachromasia.

 Composition:
It includes:
a) Albert I: Comprises of toluidine blue, malachite green, glacial acetic acid, alcohol (95%
Ethanol) and distilled water
b) Albert II: Iodine in potassium iodide

 Procedure:
- The smear is heat fixed.
- It is flooded with Albert I for 5 minutes, then the excess stain is drained out.
- Albert II is poured over the smear so as to cover it completely for 1 minute.
- Slide is washed in water, blotted dry and examined under oil immersion field.

 Interpretation:
Corynaebacterium diphtheriae appears as green coloured bacilli arranged in Chinese letter or
cuneiform pattern, with bluish black metachromatic granules at polar ends.
These can be differentiated from diphtheroides which do not show granules and are arranged in
palisade arrangement.

Albert stained smear of Corynaebacterium diphtheriae showing green bacilli with blue-black
metachromatic granules
 III) Acid fast stain:
It is also known as Ziehl-Neelsen stain, used to identify acid fast organisms or structures (e.g.-
Mycobacterium tuberculosis). Acid-fast organisms appeared bright red in the blue background.
IV) Negative stain:
A simple staining technique in which microorganisms are not stained but are made visible against
dark background. It is also known as background or indirect staining. Ex: India ink, Nigrosin,
Eosin
 Procedure:
A drop of deposits from centrifuged clinical specimen such as CSF or other fluid, is mixed on a
slide with one drop of India Ink or Nigrosin dye and overlaid with a coverslip. Then it is examined
under low power field (10x) first, then focused under high power objective (40x).

 Interpretation:
The background gets stained black whereas the unstained structures stand out in contrast.
This technique is particularly useful in visualizing the capsules of Cryptococcus neoformans in
CSF or other fluids. It may also be used to demonstrate bacterial capsules.

India ink preparation of CSF sample showing clear refractile halo surrounding round yeast cells
presenting Capsules.
 V) Impregnation stain:
Bacterial cells and structures that are too thin to be seen under the light microscope are thickened
by impregnation of silver on their surface to make them visible.
E.g.- demonstration of bacterial flagella and spirochetes
VI) Bipolar stain:
It is a staining method in which two ends of cells are more deeply coloured/ stained than the center
part of the cells.
E.g.- Wayson stain for Yersinia pestis.
VII) Simple stain:
A staining technique where a watery solution of a simple basic dye is used.
E.g.- Loeffler’s methylene blue, Polychrome methylene blue, dilute carbol fuchsin
VIII) Fluorescent stain:
It is a type of staining method in which fluorochromes bind chemically with a variety of proteins
of cells and emits fluorescent signals in visible range (490 nm-555 nm) on excitation with UV rays
or short wave length visible light. This fluorescent signal can be visualized in direct smears of
biological materials. Commonly used fluorochromes are Fluorescein isothiocyanate (FITC) and
tetramethylrhodamine isothiocyanate (TMRI).
E.g.- Auramine- rhodamine dye, Acridine orange.
2. Examination of unstained preparation:
Presence of microorganism can also be detected indirectly from unstained preparations. These are
as follows-
i) Hanging drop preparation
ii) Saline mount
iii) Iodine mount
iv) Potassium hydroxide (KOH) mount
v) Dark field microscopy

I) Hanging drop preparation:

Definition:
Motility is defined as active, unidirectional and purposeful movement of bacteria with relative
change in place and position of bacteria in that field.
Different types of demonstration of motility:
 Direct Demonstration: (Demonstration of flagella)
- Tannic acid staining (leifson staining, Ryu’s staining)
- Demonstration under dark ground, phase contrast & electron microscopy.
 Indirect Demonstration:
- Hanging drop preparation
- Unstained wet mount
- Semisolid agar media stab culture
- Cragies tube method
- U-tube method
- Swarming
- Capillary tube method
Procedure of Hanging drop preparation:

One clean coverslip is taken and white paraffin / plasticine is applied on all four sides.

One sterile loop is taken and a loopful of normal saline is placed on the centre of the coverslip.
Now, the loop is again made sterile and few bacterial colonies are taken from supplied culture
plate. The colonies are then transferred to the saline drop on the coverslip carefully so that the drop
does not spread.

A clean slide is put on the preparation and now the preparation is turned over. The drop is now
seen to be hanging from the undersurface of the coverslip. The preparation is now ready for
examination.

Examination:

The margin (air –water interface) of the drop is first focused under low power objective and it
appears as a refractive curved line. Now, the high power objective is used to observe the motility
of the organisms.

Result:

Hanging drop preparation is made from the supplied culture plate and the organism are found to
be motile.
True motility should always be differentiated from passive drifting and brownian movement.
 II) Saline mount/ Wet mount:
Purpose: To determine biologic activity of microorganisms, including motility or reactions to
certain chemicals, or serologic reactivity in specific antisera. The latter includes the Quellung
(capsular swelling) reaction used to identify different capsular types of Streptococcus pneumoniae
and Haemophilus influenzae.
Techniques: A small quantity of the specimen to be examined is dispersed into a drop of saline
on a microscope slide. A coverslip is overlaid and examined under microscope.
III) Iodine mount:
Purpose: Iodine mounts are usually used in parallel with saline mounts when examining faeces or
other materials for intestinal protozoa or helminth ova. The iodine stains the nuclei and
intracytoplasmic organelles so that they are more easily seen.
Techniques: A small amount of fecal matter or other material is mixed in a drop of the iodine
solution on a microscope slide. This is mixed to form an even suspension and a coverslip is placed
over the drop. The mount is then examined directly under microscope.
IV) Potassium hydroxide (KOH) mount:
Purpose: It is used to aid in detecting fungal elements in thick mucoid material or in specimens
containing keratinous material, such as skin scales, nails or hair. The KOH dissolves the
background keratin, unmasking the fungal elements to make them more apparent.
Techniques: Fragments of skin scales, nails or hair are suspended in a drop of 10% KOH solution.
A coverslip is overlaid and kept for half an hour at room temperature. The mount may be gently
heated to accelerate the process. Then it is examined under microscope (40x) for fungal hyphae or
spores.
V) Dark field microscopy:
Purpose: To visualize certain delicate microorganisms. This method is particularly useful in
demonstrating spirochetes Treponema pallidum.