MR. ALI MUSHTAQ Microscopical Techniques.ppt

### Discrepancies in Safe Working Practices

Ensuring safe working practices is crucial across all industries, but discrepancies often arise due to various factors, compromising worker safety and operational efficiency. These discrepancies can stem from a lack of comprehensive training, inadequate communication, and inconsistent enforcement of safety protocols. For instance, employees may receive initial safety training but lack ongoing education to address evolving risks and new equipment. This gap can lead to misunderstandings and mishandling of tools or substances, increasing the likelihood of accidents. Additionally, communication breakdowns between management and workers can hinder the reporting and addressing of potential hazards. When safety concerns are not effectively communicated, necessary preventive measures may be delayed or overlooked entirely.

Furthermore, inconsistent enforcement of safety regulations can create an environment where workers do not take safety protocols seriously. If some employees adhere to safety standards while others do not, it can lead to a culture of complacency, where the non-compliant behavior of a few undermines the efforts of the many. This inconsistency can also stem from management’s failure to regularly review and update safety procedures in line with the latest industry standards and technological advancements. As industries evolve, so do the risks; thus, safety protocols must be dynamic and adaptable to change.

Another critical factor contributing to discrepancies in safe working practices is the variance in safety culture across different organizations and sectors. Some industries may prioritize productivity over safety, leading to shortcuts and negligence. In contrast, others may implement stringent safety measures but fail to provide the necessary resources and support for their effective execution. This disparity in safety culture can result in uneven safety records and varying levels of risk exposure among workers.

To address these discrepancies, it is essential for organizations to invest in continuous training, foster open communication channels, consistently enforce safety regulations, and cultivate a robust safety culture. By doing so, they can minimize risks, protect their workforce, and enhance overall operational efficiency.

1. MICROSCOPICAL
TECHNIQUES

2. •Microscopical techniques can provide a rapid
presumptive diagnosis of an infection, e.g. pulmonary
tuberculosis using the Ziehl-Nelsen staining technique.
•Gram staining technique can indicate whether an
organism is Gram positive or Gram negative, a coccus or
bacillus.

3. EXAMINING PATHOGENS IN WET
PREPARATIONS
•The examination of wet preparations is mainly used:
•to examine specimens and cultures for motile bacteria.
•to examine c.s.f. for capsulated yeast cells.
•to examine specimens for fungi.

4. DETECTING MOTILE BACTERIA
•An organism is motile or non-motile can often assist in its
identification, e.g. most serovars of Salmonella are
motile where as Shigella species are non-motile. Vibrio
and Campylobacter species show a distinctive motility.

5. TRANSMITTED LIGHT MICROSCOPY
Technique using transmitted light microscopy:
The simplest way of examining a bacterial suspension for
motile bacteria is as follows:
Place a small drop of suspension on a slide and cover
with a cover glass. Avoid making the preparation too
thick.
Examine the preparation microscopically for motile
organisms, using the 10 X and 40 X objectives. Make
sure the iris diaphragm of the condenser is sufficiently
closed to give good contrast otherwise the organisms
will not be seen.

6. DARK-FIELD MICROSCOPY
•Other forms of microscopy such as phase contrast and
dark-field are recommended in preference to transmitted
light microscopy when examining organisms in unstained
wet preparations.
•Dark-field microscopy is required to detect Treponema
pallidum spirochaetes in specimens.

7. EXAMINING WET PREPARATIONS FOR
CAPSULATED ORGANISMS
•When cryptococcal meningitis is suspected, the
examination of c.s.f. for capsulated yeast cells is an
important investigation. Capsulated C. neoformans yeast
cells are best detected in thin preparations of sediment
from centrifuged c.s.f using India ink or dark-field
microscopy.

8. HOW TO MAKE SMEARS
•Smears should be spread evenly covering an area of
about 15–20 mm diameter on a slide.
•The techniques used to make smears from different
specimens are as follows:
•Purulent specimen: Using a sterile wire loop, make a thin
preparation. Do not centrifuge a purulent fluid, e.g. c.s.f.
containing pus cells.
•Non-purulent fluid specimen: Centrifuge the fluid and
make a smear from a drop of the well-mixed sediment.

9. •Culture: Emulsify a colony in sterile distilled water and
make a thin preparation on a slide.
•Sputum: Use a piece of clean stick to transfer and
spread purulent and caseous material on a slide. Soak
the stick in a phenol or hypochlorite disinfectant before
discarding it.
•Swabs: Roll the swab on a slide. This is particularly
important when looking for intracellular bacteria such as
N. gonorrhoeae.

10. DRYING AND FIXING SMEARS
•After making a smear, leave the slide in a safe place for
the smear to air-dry, protected from dust, flies,
cockroaches, ants, and direct sunlight. When a smear
requires urgent staining, it can be dried quickly using
gentle heat. Smears taken from in-patients and during
out-patient clinics must always be transported to the
laboratory in a covered container.

11. •The purpose of fixation is to preserve microorganisms
and to prevent smears being washed from slides during
staining. Smears are fixed by heat, alcohol, or
occasionally by other chemicals.

12. HEAT FIXATION
•This is widely used but can damage organisms and alter
their staining reactions especially when excessive heat is
used. Heat fixation also damages leucocytes and is
therefore unsuitable for fixing smears which may contain
intracellular organisms such as N. gonorrhoeae and N.
meningitidis.
•When used, heat fixation must be carried out with care.

13. The following technique is recommended:
•Allow the smear to air-dry completely.
•Rapidly pass the slide, smear uppermost, three times
through the flame of a spirit lamp or pilot flame of a
Bunsen burner.
•Allow the smear to cool before staining it.
•Microorganisms are not always killed by heat fixation,
e.g. M. tuberculosis.

14. ALCOHOL FIXATION
•This form of fixation is far less damaging to
microorganisms than heat. Cells, especially pus cells,
are also well preserved. Alcohol fixation is therefore
recommended for fixing smears when looking for Gram
negative intracellular diplococci. Alcohol fixation is more
bactericidal than heat (e.g. M. tuberculosis is rapidly
killed in sputum smears after applying 70% v/v alcohol).

15. A method of alcohol fixing smears is as follows:
•Allow the smear to air-dry completely.
Depending on the type of smear, alcohol-fix as follows:
•For the detection of intracellular Gram negative diplococci(N.
gonorrhoeaeor N. meningitidis), fix with one or two drops of
absolute methanol or ethanol.
•For the detection of other organisms, fix with one or two drops
of 70% v/v methanol or ethanol (absolute methanol can also
be used but a 70% v/v solution is adequate).
•Leave the alcohol on the smear for a minimum of 2 minutes
or until the alcohol evaporate.

16. OTHER CHEMICAL FIXATIVES
•Other chemicals are sometimes necessary to fix smears
which contain particularly dangerous organisms to
ensure all the organisms are killed, e.g. 40 g/l potassium
permanganate is recommended for fixing smears which
may contain anthrax bacilli.
•Formaldehyde vapour is sometimes recommended for
fixing smears which may contain Mycobacterium
species. Formaldehyde fixed smears, however, tend to
stain poorly and the chemical itself is toxic with an
injurious vapour.