Microbiology

Institute of Nursing Sciences KMU

BSN Year 1, Sem1 (OSPE) Checklist

S.NO Procedures

01 Microscope Operating Procedure

02 Gram staining, ZN Staining Procedure

03 Medical hand washing procedure
04 Medical Hand Scrubbing procedure
05 Demonstrate Microbiology Culture Media.(Blood
agar,MacConkey agar, Lowenstein jenson
medium, Nutrient agar,CLED agar,TSI)
06 Demonstrate Microbiology Lab Safety protocol.

07 Demonstrate preventive measure to control of

nosocomial infection.
08 Demonstrate various type of Isolation.
 Microscopy
Purpose:

 Microscopy is the technology of making very small things visible to the

human eye.
 Used in the diagnosis of many diseases.
 Educational microscope: used for research.
 In this lab, you will become familiar with the use of the light compound
microscope (particularly oil immersion microscopy) and will compare the
relative size and shape of various microorganisms.

Operating Procedure:

S. No STEPS
01 The microscope should be placed on a level bench, which
should be free of vibration.

02 The power socket to which the microscope is plugged should

not be loose or sparking.

03 The height of the microscope or chair should be adjusted in

such a way that the user’s eyes are directly on the eyepieces.

04 Place the specimen on the stage, switch to the x10 objective

and focus.

05 Turn on the power at the wall and the power switch on the
microscope.

06 Ensure the light beam diaphragm is opened.

07 Start with the light setting at low to medium.

08 Depending on the sample being examined, the condenser
may need to be adjusted.

09 Secure the slide using the arms on the microscope stages.

10 When starting to examine a slide, select the low powered(X4)

object lens first.

11 Your eye level should be just above the eye pieces then look
down the eye pieces and gently slide them together until you
see a single object.

12 Locate an area of the slide that includes part of the sample.

13 To bring the slide into focus rotate the coarse focus slowly
until the sample can be visualized clearly.

14 Once an area of interest has been identified, rotate through

the objective lenses to a higher power.

15 The oil immersion lens (and oil) is required to look at cell

detail or bacteria.

16 Rotate the objective lenses slightly leaving a space to add a

drop of immersion oil to the slide, directly over the spot of
light.

17 Look down the eyepieces and use the fine focus to bring the
image into focus.

18 Once the examination is completed, remove the slide and

clean the oil from the immersion lens. Only use lens tissue on
microscope objectives.
 Gram Staining Procedure

Purpose

 Gram stain is fundamental to the phenotypic characterization of bacteria.

 The staining procedure differentiates organisms of the domain Bacteria
according to cell wall structure.
 Gram-positive cells have a thick peptidoglycan layer and stain blue to
purple. Gram-negative cells have a thin peptidoglycan layer and stain red to
pink.

Reagents

1. Primary Stain: Crystal Violet Staining Reagent.

2. Mordant: Gram's Iodine

3. Decolorizing Agent Ethanol, 95%

4. Counterstain: Safranine

Smear Preparation.

 To stain material from a culture growing on solid media, place a loop full of
tap water on a slide, using a sterile cool loop transfer a small sample of the
colony to the drop, and emulsify.
 Allow the film to air dry.

Gram Staining Steps.

S. No STEPS

01 Flood the slide with crystal violet solution for up to one

minute. Wash off briefly with tap water. (Not over 5 second).

02 Flood slide with gram iodine solution, and allow to act for one
minute. Wash off with tap water. (Not over 5 second).

03 Flood slide with 95% alcohol for 10 second and wash off with
tap water. (Not over 5 second).
04 Drain the slide.

05 Flood slide with Safranine (counterstain) solution and allow

for 30second.wash up with tap water.

06 All slides of bacteria must be examined under the oil

immersion lens.

ZN Staining

It was first develop by Ziehl and later on modified by Neelson.so this method is
named ziehl nelson staining technique.

The main aims of this staining is to differentiate bacteria into acid fast group and
non-acid fasts groups, so this technique is also called acid fast staining.

Material required:

1. Carbol fuchsin stain.

2. Acid alcohol, 3%

3. Malachite green 5gl

ZN Staining Procedure:

01 Prepare bacterial smear on clean grease free slide,

using sterile technique.
02 Allow smear to air dry and then heat fix.

03 Cover the smear with carbol fuchsin stain.

04 Heat the stain until vapor just begins to rise (i.e. about
60c).Do not over heat.
05 Wash off the stain with clean water.

06 Cover the smear with 3%v/v acid alcohol for 5

minutes or until the smear is suffiently decolorized,
i.e. pale pink color appeared.
07 Wash well with clean water.

08 Cover the smear with malachite green stain 1-2

minutes, using the longer time when the smear is thin.
09 Wash off the stain with clean water.

10 Wipe the back of the slide clean, and place it in a

draining rack for the smear to air-dry.
11 Examine the smear microscopically, using the 100x oil
immersion.
 MEDICAL HAND WASHING

1. PURPOSES:

 To reduce the number of microorganisms on hands.

 To reduce the risk of transmission of microorganism to patient.
 To reduce the risk of cross contamination among patient.
 To reduce the risk of transmission of infectious organisms to oneself.

EQUIPMENT:

 Soap in soap dish

 Hand towel
 Tissue paper
 Small tray
PROCEDURE:

STEPS RATIONALE S U

1. Check hands for break in the For your protection.

skin.

2. File Nails short. Short nails are less likely to harbor

Microorganisms, scratch client, or

Puncture gloves.

3. Remove all jewelry (rings, Microorganism can lodge in

bangles, Wrist watch). jewelry and removal facilitates

4. Stand in front of the sink, Inside the sink is contaminated

keeping hands and uniform away
from the sink surface.

5. Turn on the water tap, using Water will not splash on uniform
towel paper. Adjust the flow.

6. Wet hands, holding the hands Water should flow from the least
lower than the elbow. contaminated to the most
contaminated area.
7. Apply soap to the hands (if a Soap cleanses by emulsifying fat
soap is used, rinse it before and oil and lowering surface
returning to the dish). tension.

8. Use friction – 1 min

 Palm to palm
 Rub right palm over left
dorsum Fraction and rubbing
 Inter lace fingers and clasp mechanically loosens and
palm to palm. removes dirt and transient
 Clasp back of fingers to bacteria.
opposing palm.
 Rub right thumb in rotational
fashion.
 Use circular movement for
wrist and back of palm and
rub them.
9. Thoroughly wash soap off and The circular actions help remove
rinse hands using firm rubbing & soap and microorganism
circular movements. mechanically.

10. Turn off tap using paper towel. Will prevent recontamination of
washed fingers.

11. Dry hands thoroughly, Moisture can easily damage and

including between fingers, promote growth of
microorganism.
Medical Hand Scrubbing