This document discusses laboratory work in microbiology, including the history and objectives of laboratory work. It describes guidelines for working with microorganisms, including their categorization into biosafety levels based on pathogenicity. It covers biosafety level practices and the engineering controls required for different biosafety levels. It also discusses specimen collection and handling for microbiology, including transport, rejection criteria, and standard precautions. Methods for clinical diagnosis in the microbiology laboratory include direct examination and culture/isolation.

1. Laboratory work, Symptoms and
Specimen Collecting and Handling

2. Laboratory :
‘The Workroom of a scientist
or
‘A place devoted to experimental
study of natural science.
•Definition:
A building equipped for scientific experiments , research,
teaching or for the manufacturing the drugs or chemical.
( Oxford Dictionary)

3. •History
The first laboratory (chemistry Laboratory) was established by Antoine Lavoisier
in 18thCentury.
• Objectives of laboratory work
To use the power of observation &
reasoning.
• To manipulate learning equipment's.
• To use reality to make learning easy & permanent.
• To make use of the scientific method.
• To use the laboratory method or procedure.

4. Work in the
Microbiology Lab
An Introduction to Principles and Practices
at
Biosafety Levels 1, 2, 3, & 4

5. Microorganism Categories
• How are microorganisms categorized?
• By genetics to show how they are related
• By tissues they infect to show how they cause disease
• By pathogenicity and communicability (also known as
their Bio Safety Level)

6. Guidelines for Microorganism
Use
• Besides federal law and regulations
other guidelines exist for the use and
control of microorganisms:
• CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories (BMBL)
• WHO (World Health Organization)
Biosafety Manual
• USDA (United States Department of
Agriculture) protocols

7. Guidelines for Microorganism
Use
The microbes are placed into 4
categories called :
Biosafety Levels (BSL 1-4)

8. BSL Labs
• Microbiology Laboratories are set up and maintained to
meet a specific containment level. The designated level
conveys information about infection potential and
engineering controls implemented to protect workers.

9. BSL Agents
1 Not known to consistently cause disease in healthy adults
2 Associated with human disease, hazard = percutaneous injury, ingestion,
mucous membrane exposure
3 Indigenous or exotic agents with potential for aerosol transmission; disease
may have serious or lethal consequences
4 Dangerous/exotic agents which pose high risk of life-threatening disease,
aerosol-transmitted lab infections; or related agents with unknown risk of
transmission
Biosafety Levels for Infectious Agents

10. BSL Practice
1 Standard Microbiological Practices
2 BSL-1 practice plus: Limited access, Biohazard warning
signs, "Sharps" precautions, Biosafety manual defining
any needed waste decontamination or medical
surveillance policies
3 BSL-2 practice plus: Controlled access, Decontamination of
all waste, Decontamination of lab clothing before
laundering.
4 BSL-3 practices plus: Clothing change before entering,
Shower on exit, All material decontaminated on exit from
facility
Recommended Biosafety Level Practices*

11. BSL
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
1 None required Open bench top & sink required
2 Primary barriers = Class I or II BioSafety
Cabinets; laboratory coats; gloves;
face protection as needed
BSL-1 plus:
• Autoclave available
3 Primary barriers = Class I or II BioSafety
Cabinets; protective lab clothing;
gloves; respiratory protection as
needed
BSL-2 plus:
• Self-closing, double-door access
• Exhausted air not recirculated
• Negative airflow into laboratory
4 Primary barriers = Class III Bio Safety
Cabinets or in combination with full-
body, air-supplied, positive pressure
suit
BSL-3 plus:
• Separate building or zone
• Dedicated supply and exhaust,
vacuum, and decon systems
Engineering Controls by Biosafety Level

12. Safety Resources

13. Biosafety Level 1
Standard Microbiological Practices
• Restrict or limit access
when working
• Prohibit eating, drinking
and smoking in the
laboratory
• Pippetting by mouth
strictly forbidden
2.3

14. Biosafety Level 1
Standard Microbiological Practices
2.3

15. Standard practices also include:
• Keep work areas uncluttered and clean
• No food in lab refrigerator
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Maintain insect & rodent control program

16. Decontamination
•Sterilization
•Disinfection

17. Decontamination
Definition
• Sterilization
The use of a physical or chemical
procedure to destroy all
microbial life, including large
numbers of highly resistant
bacterial spores.

18. • Disinfection
The use of a physical or chemical
procedure to virtually eliminate all
recognized pathogenic
microorganisms but not all microbial
forms (bacterial endospores) on
inanimate objects.
Disinfection
Definition

19. Decontamination
Methods
• Heat
• Chemical
• Radiation

20. • Types
• Moist – steam
• Dry
• Incineration
*The most effective method of
sterilization
Decontamination
Heat

21. • Types
• Liquids, i.e. Clorox,
hydrogen peroxide
• Gases, i.e. ethylene oxide
Decontamination
Chemical

22. • General Lab Use - Hypochlorite
Solutions
• Large Spills/Large Organic Load
• undiluted from bottle
• Small Spills/Virus Inactivation
• 10% - 1:9
• General Surface Disinfection
• 1% - 1:99
Decontamination
Chemical

23. In case of a spill
• Wear disposable gloves
• Cover large blood spill with paper towels and
soak with 1% (10000 ppm) of household
bleach and allow to stand for at least 5
minutes
• Small spill - wipe with paper towel soaked in
1% bleach
• Discard contaminated towels in infective
waste containers
• Wipe down the area with clean towels
soaked in a same dilution of household
bleach

24. Aseptic Technique
• First requirement for study of microbes
• pure cultures, free of other microbes
• Maintain a clean environment; work close
to the flame

25. •Microbiology Sample Collection and Handling
• Collection of specimens for culture differs in two ways from
collection of
• specimens for routine analysis:
• 1. Avoiding contamination by the organisms on the skin is essential if
• misleading results and inappropriate therapy are to be avoided.
• 2. Although any small volume can be cultured, the probability of obtaining a
• “positive” culture increases in proportion to the size of the sample obtained.
• Sub-optimal samples, whether from blood culture, throat swab
• or other samples may provide a false negative result which can result in
• the patient not receiving appropriate therapy.

26. •General specimen collection and processing issue
• Specimen Collection Successful laboratory diagnosis of a microbial infection
depends on many factors beginning with a well-collected sample. Proper
specimen selection, collection, and transport are all essential to ensure that a
specimen is representative of the disease process and minimally contaminated
with microorganisms present in adjacent tissues.
• Site and Timing Collect the sample from the correct anatomic site
.The timing of sample collection is also important. E.g, when submitting a
specimen for bacterial culture, samples should be collected before the
administration of antibiotics.

27. •Collection Techniques
Sterile technique and equipment. Sufficient volume ,After collection, the
specimen must be placed in an appropriately labeled leak-proof container.
• Requisition slip Each specimen must be accompanied by a requisition slip to
evaluate the specimen appropriately and relay the test results back to health
care provider without delay . The requisition slip should contain these
information :patient name, age, gender, identification number, location, name
of health- care provider, time and date of collection, specimen type, diagnosis,
and test(s) requested.
• Transport of Specimens
Rapid, optimally in less than 2 hours. For delays in transport, most specimens
should be refrigerated . Exceptions: blood, cerebrospinal fluid (CSF), and
specimens to be examined for anaerobes, fastidious organisms such as
Neisseria gonorrhoeae and Bordetella pertussis, and Trichomonas vaginalis, all
of which should be maintained at room temperature

28. • Specimen rejection criteria(1)
Improper transport temperature ,Improper transport container or medium
Prolonged transport time Unlabeled or mislabeled specimen Broken or cracked
container Leaking specimen
• Specimen rejection criteria(2)
Dried-out specimen, Inappropriate specimen for test requested ,Inadequate
volume Specimen in fixative (for culture),Duplicate sample in 24-hr period (for
urine, sputum, feces culture)
• Specimen rejection criteria(3)
When specimens are rejected, the health care provider is notified so that
another specimen may be properly submitted . If information on the requisition
is incomplete, laboratory personnel should ask a responsible person to provide
the information before processing the specimen further . If a specimen is
mislabeled, the sample should be recollected. Relabeling of a specimen is
acceptable only for difficult to collect specimens, such as tissue obtained during
a surgical procedure or CSF.

29. Specimen not routinely accepted for anaerobic culture(1)
Throat, nasopharyngeal, or gingival swabs ,Sputum Bronchial wash, lavage, or
brush (except when collected with a protected double lumen catheter)Gastric and
bowel contents
Specimen not routinely accepted for anaerobic culture(2)
Catheterized urine Female genital tract specimens collected through the vagina
Surface swabs of ulcers, wound, and abscesses
Standard Precautions
All specimens should be presumed to contain transmissible agents and therefore
should be collected and handled using standard precautions . Use of gloves, gown,
mask, and protective eyewear when there is a risk of coming in contact with the
specimen . In most clinical laboratories, a special area is designed for processing
clinical samples for culture.

30. Clinical Diagnosis by Microbiology Laboratory method(1)
Direct Examination Gram stain (general)acid-fast bacilli (AFB) (mycobacteria )KOH and/or
calcofluor white preparation (fungi )wet mount (parasites), etc. Other techniques for directly
examining specimens :direct fluorescent antibody stains (DFA),enzyme immunoassays (EIAs),DNA
hybridization or amplification assays, etc.
Clinical Diagnosis by Microbiology Laboratory method(2)
Isolate, Culture and Identification A combination of media types is used to isolate bacteria and fungi (include
enriched, nonselective, selective, or differential media);Viruses can only be cultured within mammalian cell.
Clinical Diagnosis by Microbiology Laboratory method(3)
Lengths of culture time Most routine bacterial cultures are incubated for 2 to 3 days . Mycobacterial and
fungal cultures are incubated for as long as 6 weeks . Viral cell cultures are incubated for varying lengths of
time depending on the specimen source and the growth rate of the viruses that are typically recovered from
that site.
Clinical Dignosis by Microbiology Laboratory method(4)
The condition of incubation35℃ for bacteria and viruses30℃ for fungi Various atmospheric conditions may be
utilized including ambient, CO2 enriched, microaerophilic and anaerobic.

31. Specific recommendations for each specimen type
Blood - specimen collection(1)
In general, blood for culture should not be obtained using an intravascular device . When performing a
venipuncture, the skin must be adequately disinfected to minimize contamination with normal skin flora .
Blood should be collected and incubated into the blood culture bottles using the same needle.
Blood - specimen collection(2)
Blood specimens should be collected before administering antimicrobial agents . Optimally, the specimen
should be collected just before a fever spike; however, practically, the specimen should be collected
immediately after the spike . For adults, 20 to 30mL of blood should be collected per venipuncture. Less blood
is required for children .
Blood - specimen collection(3)
For adult patient, two sets of cultures should be collected per febrile episode to help distinguish probable
pathogens from possible contaminants No more than four sets should be submitted in a 24-hour period .
Inoculated blood culture vials should be held at room temperature until they reach the laboratory.

32. Blood culture(1)Cultures for rapidly growing bacteria and yeast are usually incubated for 5 to 7 days . Cultures
for mycobacteria and slowly growing fungi are held for as long as 42 days . Many types of blood culture
systems are available, including both manual and auto-mated. Each system utilizes a noninvasive method
(i.e., colorimetric, fluorescent, or manometric methods for detecting CO2 or other gases) to monitor growth.
Blood culture(2)As soon as growth is detected from the blood specimen, a stain is performed (Gram, acid-
fast, or Giemsa stain) to determine the type of microorganism present . Positive stain results are considered a
critical value and called directly to the patient’s health care provider .Then the specimen should be sub-
cultured to solid media.
Culture of catheter tips
Performed to determine the source of a bacteremia . Semiquantitative catheter tip culture method :The
segment is rolled across a blood agar plate four times Cultures yielding organisms present in more than 15
CFU are considered to be significant, potentially indicating a catheter-related infection.
Cerebrospinal fluid (CSF)(1)
Cerebrospinal fluid (CSF) is submitted for microbiological analysis when meningitis or encephalitis is
suspected . For meningitis, the likely infection agent differs depending on the duration of symptoms. The
most likely bacterial agent of acute meningitis will also vary with the age of the individual and whether the
disease is community or nosocomially acquired.

33. • Cerebrospinal fluid (CSF)(2)
Most infectious cases of encephalitis are a result of viral infection, both
arthropod and non arthropod borne . Parasitic infections of the central nervous
system also occur, with varying clinical presentations.
• CSF - specimen collection
Obtained by lumbar spinal puncture Generally at least 0.5mL of CSF (smear,
culture, antigen tests )For mycobacterial culture, at least 3mL (greater volumes
increase recovery)
• CSF – transportationTransported to the laboratory promptly and processed as
soon as possible.If a delay in processing is unavoidable, the specimen should be
held at room temperature.If greater than 1.0mL of CSF is received for a given
test the fluid is centrifuged to allow the test to be performed on the concentrate
sediment
• CSF-laboratory diagnosis
Gram stain antigen testsIndia ink test (Cryptococcus neoformans )dark-field
microscopy of a concentrated specimen (Leptospires)acid-fast bacilli (AFB)
(mycobacteria )bacterial cultureyeast and fungi cultureviral culture

34. • Gastrointestinal Tract(1)
Feces, and in some cases rectal swabs, are submitted to the laboratory primarily to
determine the etiologic agent infections diarrhea or food poisoning . Feces should be
collected in a clean container with a tight lid and should not be contaminated with urine,
barium, or toilet paper. Optimally be examined within 2 hours of collection.
• Gastrointestinal Tract(2)
Rectal swabs should be placed in a tube transport system containing modified Stuart-’s
medium . Unpreserved stool specimens should be maintained at refrigerator temperature
during storage and transport.
• Gastrointestinal Tract(3)
It is becoming standard practice to reject stool specimens for bacterial culture and parasite
examination from patients who have been hospitalized longer than 3 days . For such
patients, examination for the toxins produced by Clostridium difficile is recommended.
• Gastrointestinal Tract -laboratory diagnosis
Bacterial culture selective and differential medium (Mac Conkey agar, Hektoen enteric or
xylose-lysine-desoxycholate agar ,etc)Fungal culture of stool is not recommended .Viral
culture To detect parasites, stool is examined microscopically for the presence of protozoa,
helminth eggs, and larvae.
• Genital Tract(1)Genital tract specimens are sent to the laboratory for determining the
cause of various clinical syndromes, including vulvovaginitis, bacterial vaginosis, etc. Many
specimens will be contaminated with the normal microbiota of the genital tract or skin;
therefore, the microbiologist must differentiate the normal flora from potential pathogens.

35. • Genital Tract(2)Organisms such as N. gonorrhoeae, C. trachomatis, and Haemophilus
ducreyi are always pathogenic, whereas organisms such as the Enterobacteriaceae, S. aureus,
and group B streptococci are pathogenic only in some clinical situations.
• Genital Tract -laboratory diagnosis(1)
direct Gram stain (only a few situations ) eg., gram-negative diplococci within poly-
morphonuclear leukocytes wet mount preparation of vaginal secretions clue cells :epithelial
cells covered with small coccobacillary bacteria vaginal pH normal≤4.5whiff test positive
:generation of a pungent, fishy odor on addition of 10% KOH to the specimen
• Genital Tract -laboratory diagnosis(2)
bacterial culture: depend on the source and the organisms likely to cause disease at that site
Tissue and aspirates should be plated to media capable of recovering fastidious organisms .
Specimens from the cervix, vagina, and urethra should at a minimum be evaluated for N.
gonorrhoeae and C. trachomatis by culture or a direct detection method.
• Genital Tract -laboratory diagnosis(3)
Fungal culture of female genital tract specimens is not productive . Viral culture remains the
gold standard for detection of Herpes Simplex Virus.
• Lower Respiratory Tract
primarily to determine the etiologic agent of pneumonia Specimen types : sputum
(expectorated or induced), tracheal aspirates, transtracheal aspirates, bronchial washes,
bronchial brushings, and bronchoalveolar lavage fluids . delivered promptly to the
laboratory. if delays are unavoidable , refrigerated .

36. • Lower Respiratory Tract -laboratory diagnosis
Gram-stained smear low-power magnification to determine the number of
squamous epithelial cells and/or neutrophils present oil immersion to
determine the relative amounts of organisms present. Intracellular organisms
should be specifically noted. Culture ,selective and nonselective media, In
addition, a medium capable of recovering fastidious organisms.
• Upper Respiratory Tract
Nasopharyngeal aspirates, washings, and swab specimens are primarily used
for the diagnosis of viral respiratory infections but may also be submitted to
diagnose pertussis, diphtheria, chlamydia infections, and candidiasis, as well as
identify carriers of N. meningitidis or S. aureus. Throat swab specimens are
generally collected to diagnose group A streptococcal pharyngitis or to detect
shedding of viruses such as enteroviruses, HSV, or CMV.
• Tissues procured at great expense and considerable risk to the patient;
therefore, for optimal evaluation enough material should be collected to allow
both histopathologic and microbiologic examination. After collection, tissues
should be placed in a sterile container and transported rapidly to the laboratory
to prevent drying.

37. • Tissues -laboratory diagnosis
Homogenized by mincing with a sterile scalpel, grinding with a mortar and
pestle or tissue grinder Gram stain or other stains examined for presence of
microorganisms, leukocytes, and squamous epithelial cells routine culture,
liquid medium and enriched agar medium bone marrow aspirates, in collection
tubes for the lysis centrifugation blood culture system or in a sterile container
• Urine -laboratory diagnosis
Screening urine specimens Gram stain dipstick tests that combine nitrate
reductase and leukocyte esterase Quantitative bacterial culture0.001-mLplastic
or wire calibrated loop blood and MacConkey agars
• Skin and Subcutaneous Lesions(1)
Ideally, the infected material is aspirated(remove) with a needle and syringe.
For transport, the material is expelled into sterile container that is tightly
capped and promptly delivered to the laboratory .If an aspirate(to suck liquid or
gas or to inhale in one’s lungs) cannot be obtained, swab specimens of exudate
collected from the deep portion of the lesion are acceptable. For bacterial and
fungal cultures, swabs may be placed in tube transport system containing
modified Stuart’s medium.

38. • Skin and Subcutaneous Lesions(2)
To recover anaerobes, an additional swab specimen must be collected and
placed in an anaerobic transport system . For viral culture, the specimen
(aspirate or swab) should be placed in viral transport medium and kept on ice .
If a delay in processing is unavoidable, specimens may be stored in the
refrigerator, except those for recovery of anaerobes (room temperature )
• Skin and Subcutaneous Lesions -laboratory
diagnosis
Gram-stain appropriate media for culture If detection of mycobacteria is
requested, specimens should be decontaminated and concentrated.

39. Thank you