STANDARD OPERATING PROCEDURE Procedure for Preparation and Staining of Specimens

	STANDARD OPERATING PROCEDURE
	Department: Quality Control	SOP No.: QC/MB/xxxx
Procedure for Preparation and Staining of Specimens		Effective Date:
		Review Date:
		Page No.: 1 of 5

OBJECTIVE:

1.1                 To lay down a procedure for Simple staining and Differential staining (gram staining).

2.0            SCOPE:

2.1                   This standard operating procedure is applicable for determination of bacteria and fungi in routine microbial analysis by staining technique in Pharma Private Limited.

3.0            RESPONSIBILITY:

3.1                 Microbiologist – Responsible for performing the activity.

3.2                 Section In-charge – Responsible for review the procedure and activity.

3.3                 QC Head – Responsible for implementing the procedure.

3.4                 QA Head – Overall compliance.

4.0            PROCEDURE:

4.1                   Most microorganisms are colourless and not clearly visible in the microscope. They are usually fixed and stained before observation by simple or differential staining to enhance distinction.

4.2                   Preparation and staining of specimens for bacteria

4.2.1             Living microorganisms can be directly examined through light microscope and must be fixed and stained to increase visibility.

4.2.2             Two different types of fixations are available: Heat fixation and chemical fixation.

4.2.3           In Heat fixation, fix the bacterial smear by gentle flame heating and air drying. This adequately preserves over all morphology except structures within the Cells.

4.2.4           Chemical fixation shall be used to protect fine cellular structures and the Morphology of larger and more delicate microorganisms.

4.3                 Staining Procedure:

4.3.1             Simple staining procedure:

4.3.1.1       Microorganisms are very often stained by simple staining, in which a single staining agent is used (simple staining value lies in its simplicity and ease of use).

4.3.1.2       Stain the smear with methylene blue for 1 to 2 minutes and wash the excess stain off with water and allow the slide to dry. Observe under microscope as per current version of SOP QC/MB/010 (Operation procedure for microscope).

4.3.1.3       Basic dyes like Crystal violet and methylene blue are frequently used to determine the size, shape and arrangement of bacteria.

4.3.2             Differential Staining/Gram’s staining procedure:

4.3.2.1       Gram’s staining is also called as differential staining procedure, it divides bacteria into two classes: gram-negative and gram- positive based on staining properties.

4.3.2.2       Use a sterile slide or pass one side of a clean glass slide through a flame several times. Allow the slide to cool before smearing with a specimen.

4.3.2.3       For bacterial suspensions in broth: Apply 1 loop of broth onto the cleaned    slide. Dilute heavy suspensions by applying less than one loop to a drop of sterile water or saline on the slide. Spread the fluid to form a film about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.

4.3.2.4       For bacterial colonies: Use water or saline to emulsify a colony or portion    of colony on the previously flamed side of the slide. Only a very small    amount of material from an isolated colony is needed for a gram stain (a loopful of a colony is excessive).

4.3.2.5       Air dry and heat fix the slide by passing it through a flame two or three times. DO NOT OVERHEAT the slide as protein in the specimen can coagulate and cellular morphology may appear distorted from excess heat.

4.3.2.6       Hold the slide with specimen side up, over the sink. Flood the slide with crystal violet and allow it to remain for 1 minute. Rinse the slide gently with cold tap water.

4.3.2.7       Apply Gram’s iodine (mordant) and allow it to remain for 1 minute. Rinse                            with water and add 2 to 3 drops of alcohol (95% ethanol) allow it to remain for 30 seconds. Quickly rinse with water.

4.3.2.8       Apply safranin and allow it to counterstain for 1 minute. Rinse with water until all free stain is removed. Blot (DO NOT WIPE) the slide dry with tissue paper.

4.3.2.9       Examine the smear under oil immersion. NEVER determine the morphology or staining reaction of bacteria with any objective other than oil immersion as per current version of SOP: QC/MB/010 (Operation procedure for microscope).

4.3.3             Observation:

4.3.3.1       Using the oil immersion lens, examine the smear for presence of bacterial cells. Note the Gram Stain reaction like positive or negative, morphology like cocci or bacilli, and cell arrangements like single cells, pairs, clusters, or chains of the cells seen.

4.3.3.2       Gram-positive bacteria shows dark purple colour.

4.3.3.3       Gram-negative organisms show pink or red colour.

4.4                 Staining procedure for fungi

4.4.1             The lacto phenol cotton blue wet mount preparation method is most widely used method for staining and detection of fungi, and it is simple to prepare.

4.4.2             The preparation has three components: Phenol (which kills any live organism), lactic acid (which preserves fungal structure) and cotton blue (which stains the chitin in the fungal cell walls).

4.4.3             The specimen may be examined directly using the stain (Lacto Phenol cotton Blue     Solution).

4.4.4             Place a drop of Lacto Phenol cotton Blue Solution at the center of a clean slide, remove a fragment of the fungal colony 2 to 3 mm from the colony edge using an inoculating loop.

4.4.5             Place the fragment in the drop of stain and tear gently.

4.4.6             Apply a cover slip, but do not push down or tap the cover slip (as this may dislodge the conidia from the conidiophores).

4.4.7             Examine the preparation under low & high magnification for the presence of characteristic structures.

4.4.8             Lacto Phenol cotton Blue Solution is useful in the recognition and presumptive identification of fungi.

4.4.9             Observation:

4.4.9.1       Yeast- large ovoid to spherical forms often occurring in clusters. Budding forms may be observed.

4.5                 Observations shall be recorded in Annexure – I of QC/MB/ (Operation Procedure for Microscope).

5.0            ABBREVIATIONS:

QC: Quality Control	QA: Quality Assurance
SOP: Standard Operating Procedure	%: Percentage
LPCBS: Lacto Phenol Cotton Blue Solution

6.0           ANNEXURES : NA

7.0           REFERENCES:

7.1                 A text book of Microbiology by Prescott, Harley and Klein.

Operation procedure for microscope