Microbiology Specimen Collection Procedures

ADVANCED CELL LABORATORIES
Al Tahlia, Olaya District

P.O. Box 28672 Riyadh 11323 – Kingdom of Saudi Arabia
MICROBIOLOGY STANDARD OPERATING PROCEDURES

TITLE / DESCRIPTION: INDEX NUMBER(S):
Quality Control Policies- Microbiology MICRO – 2017 - 01
EFFECTIVE DATE: APPLIES TO:
NOVEMBER 2017 Microbiology Section
1.0 TEST PRINCIPLE
To use the following specified procedure in a continuous manner in regards to

samples, reagents test procedures and results.
2.0 CLINICAL SIGNIFICANCE
To provide and ensure the efficient processing of diagnostic tests and techniques
that is accurate, reliable, reproductive and clinical relevance.
3.0 SPECIMEN TYPE
N/A
4.0 REQUIRED REAGENTS
N/A
5.0 CALIBRATION
N/A
6.0 QUALITY CONTROL
Expiration date of reagents and culture plate media.
7.0 PROCEDURAL STEPS
Index number Page 1 of 41

7.1 The samples should be checked for appropriate microbiology testing.

7.2 Proper labeling of the sample must be done including the patients name,
age, sex, nationality, file number,time and date collected,source of the
sample.
7.3 All samples must be delivered in biohazard bag with a request slip in the
separate pocket of the bag.
7.4 Samples must be taken cared of under safety precaution procedure.
7.5 Samples will be tested according to the source of specimen and according
to the test procedure.
7.6 All the examined samples will be placed in double biohazard yellow bags
and sent to medical waste for proper disposal.
7.7 Sample rejection should be done if these is no proper identification of the
sample.The sample request is not complete.The tube or container is
leaking.
7.8 All reagents must be properly labeled with name and expiry date.
Reagents must be stored at the proper temperature as indicated on
them.Reagents must not be used beyond expiry date.For every kit
opened,positive and negative controls must be done.
7.9 All the test tubes,innoculating loops,cotton swabs must be disposed
immediately.
7.10 Incubator must be checked daily for the appropriate temperature.
7.11 All culture plate media must be checked for the expiration date.
7.12 All the results will be written in the appropriate request form or result
print out.Critical result must be communicated immediately to the clinic
or to the attending physician.
8.0 CALCULATIONS
N/A
9.0 REFERENCE INTERVALS
N/A
10.0 INTERPRETATION OF RESULTS


N/A
11.0 REFERENCE
N/A
12.0 DISTRIBUTION
12.1 Bench copy available to all staff (hardcopy)
12.2 Chief Technologist / Supervisor (Soft Copy on CD)
12.3 Director (Soft copy on CD)

REVIEW AND APPROVALS Signature Date
Prepared by: VINOTH MUNISAMY
Mrs. Cherrielyn Gilay

Reviewed by:
Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director
Review / Revision Name Signature Summary of

Revision Number Changes
Date

Microbiology Specimen Collection MICRO – 2017 - 01
EFFECTIVE DATE: DISCEMINATION
1.0 TEST PRINCIPLE
Nuthodology for collection of specimens sent for microbiological investigation.
All clinical specimens must be collected under appropriate conditions according

to specimen type.Poorly collected specimen include failure to isolate the
causertive organism and overgrowth by contaminated or normal flora which can
lead to improper identification of pathogenic organism.
3.0 SPECIMEN TYPE
Any kind of specimen.

Sterile containers, sterile aerobic swab with transport media, sterile urine and
stool containers and any sterile tubes. No fixative is added to microbiology
samples.
5.0 TEST CALIBRATION
N/A
6.0 QUALITY CONTROL
N/A
7.0 PROCEDURAL TESTS
7.1Blood specimen
Blood must be withdrawned aseptically.Change the needle after
drawing the blood and putting it to the blood culture bottle.Skin
must be clean with proper disinfection to avoid specimen
contamination with skin flora.Disinfect the rubber diaphragm of the
culture bottle with the alcohol.Do not use iodine.
7.2Abscess, Aspirated Fluid
Syringe aspiration is preferable.
7.3Skin Lesions and Wounds
Swabs with transport media is used.
7.4Specimen from Throat, Nose, and Ear samples
These are taken with swabs in a transport media.
7.5Specimen from High Vaginal,Cervical,Vaginal:
These are done in the clinic and is collected thru swab with transport
media.
7.6Prostatic Fluid,Prostatic Discharge,Semen

These are collected in the clinics and is placed in a leaked proof
container.
7.7Sputum
It should be placed in a sterile leaked proof container.
7.8Stool Specimen


Stool should be collected in a sterile container.For suspected Shigella,
stool should be freshly submitted to the laboratory and should be
cultured immediately within 1 hour from receiving in the lab.
7.9Urine Specimen
Proper collection of urine is very important to get the accurate urine
results.Never collect urine from bed pan or urinals.Early morning
midstream advisable. Urine specimen should be submitted to the
laboratory within 2 hours of collection.If there is a delay in
transport,urine must be refrigerated.
8.0 CALCULATIONS
N/A
N/A
10.0 INTERPRETATION OF RESULT
Specimen for microbiological examination must be collected in a sterile

container or in a transport swabs with media.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION

Prepared by: Hermeliza Munoz

Reviewed by:
Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director



Date
Turn Around Time (TAT) of Tests Done MICRO – 2017 - 01
1.0 TEST PRINCIPLE


Microbiology tests will be done and reported within the approved turn around
times as maintained.
To ensure time lines of results for prompt medical decision making.
3.0 SPECIMEN TYPE
All kinds of sample.
N/A
5.0 TEST CALIBRATOR
N/A
6.0 QUALITY CONTROL
N/A
7.1 The lab staff will ensure that all tests are performed and reported within the
prescribed
turn around time.
7.2 List of test for the turn around time.

1.AFB = 24 hours
2.Gram Staining =24 hours
3.Wet Preparation=24 hours
4.KOH Preparation=24 hours
5.Routine Culture=48-72 hours
6.Blood Culture=7 days
7.Fungal Cultures=21 days
8.0 CALCULATIONS
N/A
N/A
N/A
11.0 REFERENCE
N/A
12.0 DISTRIBUTION


Reviewed by:
Chief Technologist
Approved by:


Dr. Mustafa Hameem
Director

Date

Basic Guidelines for the Identification of MICRO – 2017 - 01

Medically Important Bacteria
1.0 TEST PRINCIPLE
Helps on the identification of medically important bacteria.
Any kind of pathogenic growth.
3.0 SPECIMEN TYPE
All samples taken from the clinic with transport media such as vaginal
swabs,cervical swabs,high vaginal swabs,throat swabs,eye swabs,nasal
swabs,pus. Urine,prostatic fluid and other discharge too.
Serological commercially prepared reagents.
N/A
6.0 QUALITY CONTROL
Use reagents before the expiry date.

7.0 PROCEDURE
7.1 MICROSCOPY
Identification of bacteria is primarily knowing if it is in the group of bacilli
or cocci by doing gram staining.
7.1.1 COCCI
Cocci has two categories. The staphylococci and Streptococci. For
identifying this type of organism, we will do catalase test using 20
vol.Hydrogen Peroxide. The formation of bubbles is a positive for
catalase which is known to be Staphylococci. Coagulase negative
organisms are the Streptococci.
7.1.1.1 STAPHYLOCOCCI GRAM

Positive cocci in clusters and are catalase positive organisms. These
can be classified as Staphylococcus coagulase positive cocci as
Staphylococcus Aureus. Coagulase negative Staphylococci are the
Staphylococcus Saprophyticus and Epidermidis.
7.1.1.2 STREPTOCOCCUS,GRAM POSITIVE COCCI

Some in chain and in pairs. Some posses beta-hemolysis
(Streptococcus Pyogenes), and others has alpha hemolytic property.
They can be distinguished thru Streptococcal typing as Group A, B, C,
D, F,G.
7.1.1.3 NEISSERIA, GRAM-NEGATIVE COCCI and appears in pairs.
7.1.2 BACILLI
These are the rod-shaped bacteria that could be distinguished by the gram
stain reaction.
7.1.2.1 GRAM- NEGATIVE BACILLI


These are bacilli which takes up the color of the counter stain
(Safranin) as they appear as rod-shaped.These are Pseudomonas and
the family of Enterobacteriacae such as E.coli ,Salmonella,
Shigella,Enterobacter,Citrobacter.
7.1.2.2 GRAM-POSITIVE BACILLI

These are organisms as not takethe counter stain. They appear as rod-
shaped organisms.Lactobacillus are usually in chain.
7.1.2.3 COCCO-BACILLI
Some bacteria are pleomorphic cocci-bacilli represented by
Haemophilus specie.
7.1.3 CULTURAL CHARACTERISTICS

The appearance of colonial growth on the surface of solid media is often
very characteristic, taking note of the diameter of the colonies,their
outline,elevation translucency and colour. Changes brought about in the
medium. Example: hemolysis on the blood agar medium may also be
significant.
7.1.4 SENSITIVITY
Sensitivity of identified pathogenic organisms done. Each sample specimen
has its own specific antiobiotic dics, incubated and read according to the
zone of inhibition as to the sensitivity.
8.0 CALCULATIONS
N/A
N/A


The identification of the pathologic organisms must be reported together with
the sensitivity report. Organisms which are normal flora must be reported as
No pathogenic isolated.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION


Reviewed by:
Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director


Date
Gram Staining MICRO – 2017 - 01

1.0 TEST PRINCIPLES
Bacteria are divided into two categories,depending on whether they can be

decolorized with acetone after staining with crystal violet and treating with
Iodine.Gram Stain is used to differentiate between Gram Positive and Gram
Negative bacteria.
For the identification of Gram Positive and Gram Negative bacteria.
3.0 SPECIMEN TYPE
All kinds of samples.
4.1 Gram Crystal Violet ( Methyl Violet ) 0.5 %

4.2 Gram Iodine (Lugol’s Iodine )
- Gram Potassium Iodide 2.0 %
- Gram Resublimed I
4.3 Gram Decolorizer
- Gram Methanol 80 %
- Gram Acetone 20 %
4.4 Gram Safranine Stain ( Counter Stain ) 1.0 %
N/A
6.0 QUALITY CONTROL
N/A

7.1 Stain heat fixed smears with reagent I (Crystal Violet) for 1 minute.
7.2 Wash off with tap water and flood slide with reagent 2 (Iodine) for 2
minutes.
7.3 Wash off with tap water and decolorize. Smear with reagent 3 (Decolorizer)
for a few seconds only.
7.4 Wash well in water and counter stain with reagent 4 (Safranine) for 2
minutes.
7.5 Wash with tap water and allow to dry.
7.6 Examine under microscope using oil immersion objective.
8.0 CALCULATIONS
N/A
N/A
Gram Positive bacteria appears as Blue/Black.

Gram Negative bacteria appears as Red
11.0 REFERENCE
Gram C. (1884) Fortch.Med.2185-189.
6.0 DISTRIBUTION


Reviewed by:
Chief Technologist
Approved by:
Dr. Mustafa Hameem


Director

Date

TB Ziehl-Neelsen Stain MICRO – 2017 - 01
1.0 TEST PRINCIPLES
The Ziehl Neelsen procedure differentiates the acid-fast microorganisms. These

organisms do not loose the Funchine Stain when are decolorized with alcohol
acid and remain red-pink coloured.
The determination of mycobacterium is done on Ziehl- Neelsen Stain. It is a rod

shape bacilli that stains red on pale blue background.
3.0 SPECIMEN TYPE
Sputum, Urine, Bronchial Aspirate.
4.1 Carbol Funchin

Basic Funchin 1.5 %
Basic Funchin 4.5 %
Phenol 15 %
Ethanol 15 %
Tensoactives 15 %


Moisturizer 15 %
4.2 TB Decolorizer
Ethanol 97 %
HCC 3%
4.3 Methylene Blue

Methylene Blue 0.45 %
Ethanol 35 %
N/A
6.0 QUALITY CONTROL
Reagents must be used until the expiry date.
7.1 Prepare a thin smear and heat-fix by gently heating in the flame.
7.2 Flood fixed smear with Carbol Funchin and let it stand for 10 minutes.
Do not heat.
7.3 After this period, add some of the decolorizer washing immediately
afterwards with water. Repeat this operation until smear becomes pale
pink.
7.4 Finally cover the slide with methylene blue for 5 minutes.
7.5 Rinse with water and air dry.
7.6 Examine under oil immersion objective
8.0 CALCULATIONS
N/A

N/A
Acid alcohol resistant mycobacteria are seen as red on pale blue background.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION


Reviewed by:
Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director

Date

Semen Analysis MICRO – 2017 - 01
1.0 TEST PRINCIPLE
Semen Examination usually carried out as a part of the investigation of

infertility and is required.
For the assessment of fertility of men in cases of infertility and delayed

pregnancy in women.
3.0 SPECIMEN TYPE
Semen


10 % Formalin
N/A
6.0 QUALITY CONTROL
N/A
7.1 SAMPLE COLLECTION

7.1.1 The patient is instructed to provide a semen specimen after 3 days of
sexual
abstinence.
7.1.2 Sample is collected in a clean, leak-proof, dry container with a wide
opening.
7.1.3 Patient should be instructed that no water, soap,creams or any other
substance should be allowed to come in contact with the specimen.
7.1.4 Specimen collection time is marked.
7.1.5 It is preferably necessary to collect semen sample in the laboratory to
ensure proper time of liquefaction.
7.1.6 Safe handling of specimen must be strictly observe as potentially
infective and could transmit HIV and hepa viruses.
7.2 PHYSICAL EXAMINATION

7.2.1 VOLUME


The volume of the ejaculated semen should be measured with normal
volume of 2.5 ml. If semen culture is requested, it should be the first
thing to be done in order to avoid contamination.
7.2.2 APPEARANCE
Samples should be examined immediately after ejaculation, normally,
it is homogenous,grayish white appearance.Viscousity can be
performed by gentle aspiration of the liquefied semen and let it drop
from the pipette by the gravity. Normally it leaves in the discrete drops.
Abnormal viscousity, semen drops in threads more than 2 cm long.It
interferes with sperm counting abnormality of the semen.
7.2.3 LIQUEFACTION TIME
Normal semen liquefies within 60 minutes at room temperature.
7.2.4 REACTION (Ph) is determined using litmus paper. The normal pH is
in the range of 7-8 which is alkaline pH less than 7 in cases of
azoospermia.
7.3 MICROSCOPIC EXAMINATION

7.3.1 Examine microscopically a drop of a well-mixed semen specimen
under high power field microscope. Check for the motility,abnormal
sperm cells,Pus, RBC’s,Epithelial cells,spermatogenic clumping of cells
under high power field microscope.
8.0 CALCULATIONS
( 1:20 )
Total number of semen counted = number of semen counted in 4 large squares
x 50,000
( 1:10 )
Total number of sperm = number of spermatozoa counted in 4 large squares x
100,000
N/A

Normal sperm count is 20 million/cc or more. Patients with zero sperm is

known as azoospermia.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION

Reviewed by:
Chief Technologist
Approved by:
Dr. Mustafa Hameem
Director


Date


Urine Analysis MICRO – 2017 - 01
1.0 TEST PRINCIPLE
Examination for the upper respiratory tract infection.
The examination of urine through chemical reaction and microscopic

examination.
3.0 SPECIMEN TYPE
Urine
Uritop + 10 Strips
N/A
6.0 QUALITY CONTROL
N/A


7.1 Fill up urine in test tube
7.2 Check for the chemical reactions using a Uritop + 10 strips.
7.3 Compose the chemical reaction thru the color chart.
7.4 Centrifuge the urine for 5 minutes at 3,000 rpm.
7.5 Discard the supernatant and read the sediment under the low and high power
objective.
8.0 CALCULATIONS
N/A
N/A
Chemical reactions are done using the Uritop Strips by comparing the color
thru the color chart as to their specific Gravity,pH,
Blood,Leukocytes,Glucose,Urobilinogen and Protein. Microscopic
examination is done under the low and high power objective for the Pus,
RBC,Epithelial cells, Casts, Crystals and other parasites.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION




Reviewed by:
Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director

Date

Stool Analysis MICRO – 2017 - 01

1.0 TEST PRINCIPLE
Examination of stool samples for parasitic infection.
Examination of stool sample must be done immediately upon receiving the

samples for any parasites.
3.0 SPECIMEN TYPE
Stool
Normal Saline Solution
N/A
6.0 QUALITY CONTROL
N/A
7.1 Examine the stool for its color and consistency.

7.2 In a saline solution, make a smear of the stool.
7.3 Examine in the microscope under low power objective for any parasitic ova
and undigested food particles. High power objective for Pus, RBC’s, Fat
Globules, Motile Parasites such as Amoebae and Flagellates.


8.0 CALCULATIONS
N/A
N/A
Microscopic examination of stool for parasitic infection is being reported.
11.0 REFERENCE
N/A
12.0 DISTRIBUTION

Reviewed by:


Chief Technologist
Dr. Mustafa Hameem

Approved by:
Director

Date

Microbiology Specimen Collection Procedures

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ADVANCED CELL LABORATORIES

Al Tahlia, Olaya District

MICROBIOLOGY STANDARD OPERATING PROCEDURES

Quality Control Policies- Microbiology MICRO – 2017 - 01

EFFECTIVE DATE: APPLIES TO:

NOVEMBER 2017 Microbiology Section

1.0 TEST PRINCIPLE

To use the following specified procedure in a continuous manner in regards to

2.0 CLINICAL SIGNIFICANCE

3.0 SPECIMEN TYPE

4.0 REQUIRED REAGENTS

6.0 QUALITY CONTROL

Expiration date of reagents and culture plate media.

7.0 PROCEDURAL STEPS

Index number Page 1 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

7.1 The samples should be checked for appropriate microbiology testing.

9.0 REFERENCE INTERVALS

10.0 INTERPRETATION OF RESULTS

Index number Page 2 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

12.1 Bench copy available to all staff (hardcopy)

12.2 Chief Technologist / Supervisor (Soft Copy on CD)

12.3 Director (Soft copy on CD)

Index number Page 3 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

REVIEW AND APPROVALS Signature Date

Prepared by: VINOTH MUNISAMY

Mrs. Cherrielyn Gilay

Dr. Mustafa Hameem

Review / Revision Name Signature Summary of

Index number Page 4 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

TITLE / DESCRIPTION: INDEX NUMBER(S):

Microbiology Specimen Collection MICRO – 2017 - 01

EFFECTIVE DATE: DISCEMINATION

NOVEMBER 2017 Microbiology Section

1.0 TEST PRINCIPLE

Nuthodology for collection of specimens sent for microbiological investigation.

2.0 CLINICAL SIGNIFICANCE

All clinical specimens must be collected under appropriate conditions according

3.0 SPECIMEN TYPE

Any kind of specimen.

4.0 REQUIRED REAGENTS

Index number Page 5 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

5.0 TEST CALIBRATION

6.0 QUALITY CONTROL

7.0 PROCEDURAL TESTS

7.6Prostatic Fluid,Prostatic Discharge,Semen

Index number Page 6 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

9.0 REFERENCE INTERVALS

10.0 INTERPRETATION OF RESULT

Specimen for microbiological examination must be collected in a sterile

12.1 Bench copy available to all staff (hardcopy)

12.2 Chief Technologist / Supervisor (Soft Copy on CD)

12.3 Director (Soft copy on CD)

Index number Page 7 of 41

MICROBIOLOGY STANDARD OPERATING PROCEDURES

REVIEW AND APPROVALS Signature Date

Prepared by: Hermeliza Munoz

Mrs. Cherrielyn Gilay