Introduction to Microbiology

MICROBIOLOGY
➢ Study of organisms that are
too small to be seen by the
naked eye.

➢ PURPOSE:
responsible for the
identification of
pathogenic
microorganisms and for
hospital infection control.
HISTORY OF MICROBIOLOGY
LUCRETIUS & ▪ Suggested that diseases were caused by
GIROLAMO FRACASTORO “INVISIBLE LIVING CREATURES”
▪ “ FIRST TRUE MICROBIOLOGIST”
▪ First person to observe and accurately describe
ANTON VAN
living microorganisms (such as bacteria and
LEEUWENHOEK
parasite)
▪ “ANIMALCULES”
▪ Proposed the use of heat in killing
LOUIS PASTEUR
microorganisms (aseptic technique)
IGNAZ SEMMELWEIS ▪ “ FATHER OF HANDWASHING PROCEDURE”
▪ Introduced the system of antiseptic surgery.
JOSEPH LISTER
▪ Used PHENOL as anti-microbial agent
GERM THEORY OF DISEASE
ROBERT KOCH
KOCH’S POSTULATES
✓The microorganism must be present in every case of the disease but absent from a
healthy host.
✓The suspected microorganism must be isolated from a diseased host and grown in
a pure culture.
✓The same disease must be present when the isolated microorganism is inoculated
into a healthy host.
✓The same organism must be isolated again from the diseased host.
MICROBIOLOGY
In large laboratories,
the section may be
divided into
Bacteriology,
Mycology,
Parasitology, and
Virology.
The microbiology section is
responsible for the
identification of
pathogenic
microorganisms and for
hospital infection control.
In large laboratories, the
section may be divided
into BACTERIOLOGY,
MYCOLOGY,
PARASITOLOGY,
VIROLOGY.
Sample Collection and Handling
Phlebotomists are responsible
for collecting blood cultures and
may be required to obtain throat
cultures (TCs) and instruct
patients in the procedure for
collecting urine samples for
culture. Specific sterile
techniques must be observed in
the collection of culture samples
to prevent bacterial
contamination.
MYCOLOGY
Fungi are
identified
primarily by
culture
growth and
microscopic
morphology.
Mycology Culture Considerations:
1. Fungal cultures are incubated at 30°C.
2. Growth requires from several days to several weeks.
3. Cultures should be maintained in a high-humidity
environment.
4. Several techniques are used to obtain culture material for slide
preparation.
A. Tease mount method
B. Cellophane tape method
C. The slide culture method uses a block of agar overlaid
with a cover slip.
 BODY SITES AND POSSIBLE FUNGAL PATHOGENS
Blood Candida spp.,Cryptococcus neoformans
Cerebrospinal Fluid Cryptococcus neoformans
Microsporum
Hair, Nail, Skin Epidermophyton
Trichophyton
Candida albicans
Lungs Aspergillus
Histoplasma capsulatum
Urine/Genital Tract Candida albicans
VIROLOGY
Viruses must be
cultured in living
cells, and most
laboratories send
viral specimens for
culturing to
specialized
reference
laboratories.
Specimen Processing for Diagnosing Viral Diseases
1. Samples should generally come from the infected site.
a. Skin: Rash site and, depending on the virus, serum and urine
b. Respiratory: Sputum or throat swabs
c. CNS (e.g., meningitis and encephalitis): For diagnosis of meningitis,
cerebrospinal fluid (CSF) and serum, as well as stool or throat
swabs, can be collected because viruses are sometimes shed into
these sites.
d. Urogenital: Needle aspirates and endocervical and urethral swabs
e. Gastrointestinal tract: Stool samples and rectal swabs
f. Eye infections: Eye swabs and corneal scrapings
Sample Transport

1. Samples for viral culture must be placed into a viral transport

medium (VTM).
2. VTM contains: (a) Buffered saline (b) Protein stabilizers
(c). Antimicrobials that inhibit bacterial and fungal growth
3.Samples for viral cultures can be refrigerated in VTM for
about 48 hours, but they should never be frozen at -20°C.
4.Samples can be stored at -70°C; however, infectivity will be
diminished.
 BODY SITES AND POSSIBLE VIRAL AGENTS
Influenza Virus, Parainfluenza Virus, Respiratory
Respiratory System Syncytial Virus (RSV), Epstein-barr Virus (EBV),
Coronavirus, Adenovirus
Enterovirus, Echovirus, Herpes Simplex Virus Type
Viral Meningitis
1 (HSV-1), HSV-2
HSV-1, HSV-2, VZV, echovirus, measles virus,
Cutaneous Infections
rubella virus
Genital Infections HSV-2 and human papillomavirus
Gastroenteritis Rotaviruses, Norwalk viruses, Adenoviruses
Eye Infections HSV, adenovirus
Neonatal Infections HSV, CMV, and Rubella Virus
VIRAL IDENTIFICATION: Histology and Cytology
1. Cellular inclusions are diagnostic for many viruses.
2. Most DNA viruses replicate in the nucleus, they
often produce nuclear inclusions.
3. RNA viruses produce cytoplasmic inclusions (assembled in
the cytoplasm).
4. HSV and VZV cause intra-nuclear inclusions.
5. CMV induces enlarged (cytomegalic) cells with a basophilic
intranuclear inclusion referred to as "owl eye" inclusion.
VIRAL IDENTIFICATION: Viral Isolation

1. Cell Culture
a. Primary Cell Cultures (Monkey Kidney Cells)
b. Established Cell Lines (WI-38, MRC-5, IMR-90).
c. Continuous Cell Lines (HeLa, HEp-2, A549, Vero cells).
Slides are made from infected cell cultures and examined for
cellular changes, including clumping, vacuoles, inclusions, granules,
cell fusion (i.e., syncytium—multinucleated cell development), and
cellular destruction. These visible changes are referred to as
cytopathic effect (CPE).
CYTOPATHIC EFFECT
VIRAL IDENTIFICATION: Viral Isolation

2. Embryonated Eggs (Growth of Virus)

3. Animal Models (used in research studies).
4. Electron Microscopy (Due to size, most individual virions can
only be seen by electron microscopy).
5. Viral gene probes and nucleic acid amplification (Polymerase
Chain Reaction [PCR]).
6. Detection of viral antigen and host antibody against
specific virus.
BACTERIOLOGY
Identification of
bacteria is based on
morphology,
Gram stain reactions,
oxygen and
nutritional
requirements,
and biochemical
reactions.
General Guidelines for Specimen Collection:
▪ Specimens should be collected during the acute or early phases
.
of an illness.
▪ The correct anatomic site must be identified/selected for collection of
the specimen.
▪ The site of the infection should be cleansed properly before collection.
▪ The quantity of the collected specimen should be sufficient for
diagnostic testing.
▪ If possible, specimens should be collected before antibiotics are
administered.
General Guidelines for Specimen Collection:
▪ Swabs are generally poor specimens (Except nares and throat specimens)
compared
.
with tissues or needle aspirates.
▪ For lesions, wounds and abscesses, the specimen is collected from the advancing
margin of the lesion preferably by needle aspiration rather than by swabs.
▪ Aspirated specimens must be placed into a sterile tube or transport vial and not
squirted onto a swab.
▪ For patient-collected specimen, clear instructions should be given by an
authorized healthcare personnel, and it should be never be assumed that the
patients knows how to collect a specimen.
▪ The specimen must be labelled accurately with the specific anatomic site and the
patient demographic profile.
.
Specimen Container:
1. Specimens
. for microbiology cultures should be in sterile containers.
▪ Stool specimen can be collected IN CLEAN, NON-STERILE
CONTAINER.
2. Swabs are used for specimens from the upper respiratory tract,
external ear, eye and genital tract.
▪ Swabs are not recommended for routine collection.
▪ If swabs are used, Dacron or calcium alginate swabs are preferred
rather than cotton-tipped ones.
Specimens Used for Microbiologic Study:
▪ Most microbiology samples are obtained from the:
Blood
Urine
Throat
Sputum
Genitourinary tract
Wounds
Cerebrospinal fluid
Feces
Specimens Used for Bacteriologic Study:
Blood
▪ The venipuncture site should be disinfected with 70%
alcohol and betadine.
▪ To ensure a complete antisepsis, betadine should be in
contact with the skin for 30 to 60 seconds.
▪ Blood should be drawn during the time of febrile
episode.
▪ For the manner of collection, two sets should be drawn, from the
same drawn.
▪ No more than three sets should be drawn in a 24-hour period.
▪ Aerobic and anaerobic culture bottles are used.
Specimens Used for Bacteriologic Study:
Urine (Clean-catch voided Midstream/ Clean-voided
Specimen or CVS)
▪ The first morning urine is also preferred because its
provides a more concentrated specimen.
▪ A pure culture of E. coli that 105 CFU/ml represents a
true infection.
▪ For females, the area should be cleaned first soap and
water, and the labia should be held apart to begin
voiding.
▪ For males, the glands should be cleaned with soap and
water; the foreskin should be retracted if necessary.
Urine (Straight Catheter)
▪ After inserting the catheter into the bladder, the first
15 ml of the urine should be voided or expelled by the
bladder before collecting the ensuing urinary stream as
specimen.
Urine (Indwelling Catheter)
▪ The catheter collection port should be cleaned before
starting the collection procedure.
▪ 5 ml to 10 ml of urine should be aspirated with needle and
syringe.

Urine (Suprapubic Aspirate)

▪ The skin should be disinfected prior to the specimen collection.
▪ The authorized healthcare personnel should aspirate using a needle above the
symphysis pubis through the abdominal wall into the full bladder.
Specimens Used for Bacteriologic Study:
Respiratory Tract (LRT: BAL, Bronchial Brush, Bronchial
Wash)
▪ An anaerobic culture is appropriate
Respiratory Tract (Sputum)
▪ Prior to the collection, the patient should rinse his/her mouth w/ sterile H2O.
▪ A first-morning specimen is preferred for an acid-fast bacilli
microscopy.
▪ For mycobacterial infection, two or three consecutive early morning sputum
specimens should be submitted.
▪ The methods of collecting sputum can be through deep cough
(expectorated sputum), or aerosol-induced (induced sputum) for
pediatric and uncooperative patients.
Specimens Used for Bacteriologic Study:
Respiratory Tract (Nasopharyngeal Specimen)
▪ It is the specimen of choice for the recovery of Bordetella
pertussis.
▪ For the procedure, a flexible swab is inserted through the
nose into the posterior nasopharynx and rotated for five
seconds.

Respiratory Tract (Pharyngeal/Throat Specimen)

▪ It is the recommended specimen for the routine culture
of group A streptococci.
▪ For the procedure, the posterior pharynx and tonsil should
be without touching the roof and sides of the mouth and
tongue.
Specimens Used for Bacteriologic Study:
Genital Tract (Bartholin Cyst)
▪ The skin should be disinfected before collecting
specimen.
▪ The specimen should be aspirated.

Genital Tract(Cervix)
▪ The mucus should be removed before collecting the
specimen.
▪ A lubricant should not be used on the speculum and
the cervix should be thoroughly swabbed.
Genital Tract (Urethra)
▪ A flexible swab should be inserted two to four centimeters into the urethra and
rotated for two seconds or alternatively, some discharge can be collected on a
JEMBEC transport system.
▪ The swab must be moistened with Stuart’s or Amies medium.

Genital Tract(Vagina)
▪ Exudates should be removed before specimen collection.
▪ The swab must be moistened with Stuart’s or Amies medium.
▪ The secretion from the mucous membrane of the vagina are swabbed.

Genital Tract (Prostate Glands)

▪ The gland should be cleaned with soap and water.
▪ The swab must be moistened with Stuart’s or Amies medium.
▪ The secretion on the swab or in the tube should be collected.
Specimens Used for Bacteriologic Study:
Abscess (Lesions, Wound, Pustule, Ulcer)

▪ It is preferable to use a needle and syringe in

collecting specimens to aspirate the drainage.
▪ Needle aspiration enhances the recovery of
anaerobic bacteria.
▪ There are two types of abscess:
(1) Superficial Abscess
(2) Deep Abscess
Specimens Used for Bacteriologic Study:
Body Fluid (Amniotic, Ascites/peritoneal, Joint/Synovial, Pericardial, and
Pleural)

▪ The skin should be disinfected before aspirating the sample (needle

aspiration).
▪ Concentrating the specimen through centrifugation or filtration may
be required prior to smearing and cultivation.
▪ The sample should be inoculated as soon as it is received.
Specimens Used for Bacteriologic Study:
Cerebrospinal Fluid
▪ The skin should be disinfected before aspirating the sample
▪ Rapid diagnostic testing like gram staining and
cryptococcal antigen test is the recommended method
for this specimen.
▪ The required volume is 10 ml (microbiology and chemical
tests).
▪ The storage should be six hours at 35 degree Celsius.
▪ Bacteria associated with meningitis (Neisseria
meningitides, Haemophilus influenza) are fastidious and
susceptible to cold temperature or drying.
▪ Gram stain can be performed by cytocentrifugation.
Specimens Used for Bacteriologic Study:
Eye (Conjunctiva)
▪ Samples should be obtained from both eyes using two
separate swabs.
Eye (Corneal Scraping)
▪ An aerobic swab that is moistened with Stuart’s
or Amies medium or pre-moistened with sterile
saline may be utilized.
▪ Samples should be obtained from both eyes
using two separate swabs.
Specimens Used for Bacteriologic Study:
Gastrointestinal Tract (Gastric Biopsy)
▪ The specimen is recommended for the detection and isolation of Helicobacter
pylori.
Gastrointestinal Tract (Rectal Swab)
▪ For microscopy, methylene blue is utilized to observe fecal leukocyte.
▪ For the collection of specimen, a 2.5-cm swab is inserted through the anal sphincter.
Gastrointestinal Tract (Stool Culture)
▪ For out-patient, three specimens are collected every other day.
▪ For in-patients, three specimens are collected every day.
▪ If a patient has received anti-parasitic drugs, the specimen collection should be done
after 7 to 10 days.
▪ For microscopy, methylene blue is utilized to observe fecal leukocyte.
Specimens Used for Bacteriologic Study:
Foreign Bodies (IUD)
▪ It is usually cultured for the detection of Actinomyces species.
▪ For the procedure, the skin is disinfected before the removal of the IUD.

Foreign Bodies (Catheters, Pins, Prosthetic Valves)

▪ A segment of the catheter (5 cm to 7 cm) is rolled four times across the
agar using a sterile forceps (Maki roll technique).
▪ When using catheters and valves, more than 15 colonies are required
to perform identification and susceptibility tests.
▪ The whole Foley catheter should not be cultured.
Specimens Preservation:

▪ If the
. transport of the specimen to the laboratory or its processing is
delayed the specimen can be maintained with the use of preservatives,
holding media or even a culture media.

▪ Stool specimen for Clostridium difficile toxin assay should be collected

without a preservative and can be refrigerated, but if the specimen
cannot be processed within 48 hours, it should be stored at - 70 ̊C.
Specimens Preservation:
PRESERVATIVES
▪ . Boric acid maintains the appropriate colony counts for urine specimens
at room temperature for 24 hours.
▪ Preservatives should not be added on fecal specimen.

TRANSPORT/ HOLDING MEDIA

▪ These media maintain the viability of the microorganism present in a
specimen but do not allow their multiplication.
▪ Charcoal is sometimes added to these media to absorb fatty acids
present in the specimen and could kill fastidious organism like
Neisseria gonorrhoeae and Bordetella pertussis.
Specimens Preservation:
ANTICOAGULANTS
.

▪ These are used to prevent clotting of specimen including blood, bone

marrow and synovial fluid.
▪ 0.025% sodium polyanethol sulfonate (SPS) is used for a blood
culture media because Neisseria species and some anaerobic bacteria
are sensitive to higher concentration.
▪ Synovial and peritoneal fluids can also be inoculated into a blood
culture broth media.
Specimens Preservation:
ANTICOAGULANTS
.

▪ The reasons for using 0.025% SPS as the preferred coagulant for blood
culture are as follows:

a. to inhibit phagocytosis and complement activation

b. to neutralize antibiotics
c. to neutralize the bactericidal effect of plasma
Specimens Storage:
Ambient
Refrigerator Temp. Incubator Freezer Temp.
. Room Temp.
(4 C
̊ ) Temp. (35 C
̊ ) (-20 to -70 C
̊ )
(22 to 25 ̊C)
▪ Urine, Stool, Sputa, ▪ CSF ▪ Anaerobic ▪ Sera for serological
Swabs, ▪ Recommended culture, sterile studies
Viral Specimen preservation body fluid ▪ Tissues or
▪ Anaerobic bacteria temp. for genital specimen that are
should never be samples specimen, eye to be stored for a
stored in ref. temp. intended for and ear swab. long time.
▪ Stool isolation of C. gram staining,
difficile toxin can be culture and
stored up to 3 days. sensitivity.
Storage Requirements Prior to Specimen Process
Plate as soon as 24 hours at room Indefinitely Immediately at
.
possible temperature at RT 35 C
̊
▪ Body fluid ▪ Abscess ▪ Hair, nail, ▪ Blood
▪ Bone specimen ▪ Outer ear specimens skin scraping ▪ Bone marrow
▪ Gastric biopsy ▪ Eye and conjunctiva ▪ Cornel scraping
▪ Suprapubic specimen
aspirate of urine ▪ Genital tract
▪ Prostatic specimen
specimen ▪ URT specimen
▪ Tissue
Storage Requirements Prior to Specimen Process
Six hours at 35 C
̊ 24 hours at 4 ̊C 72 hours at 4 ̊C
.
▪ CSF (although, ▪ LRT specimen ▪ Rectal swab
immediate processing is ▪ Clean-voided midstream urine ▪ Stool culture
recommended) ▪ Straight catheter urine
▪ Indwelling catheter urine
▪ Foreign devices
Specimens Transport:
1. Ideally specimen should be transported to the laboratory immediately
and
.
preferably within 30 minutes of collection and not longer than two
hours.
▪ Specimens should be transported and maintained as much as possible to
its original state when it was collected.
▪ For anaerobic bacteria, the transport should not take more than 10
minutes.
▪ For CSF samples, the transport should be done immediately and
preferably within 15 minutes after collection.
▪ Change in oxygen, pH, and temperature caused by prolonged transport
can prevent the recovery of certain microorganism and allow the
overgrowth of other.
Specimens Transport:
2. All specimen container should be leak-proof.
3. All
.
specimen should be transported to the laboratory immediately after
collection.
▪ Any delay may decrease the number of pathogens and hasten the
multiplication of indigenous microbiota.
4. All specimen containers should be placed in sealable, leak proof plastic
bags.
▪ Patients specimen or culture isolates must be triple-packed before
being shipped.
▪ Specimen bags should be marked with a biohazard label.
 Transport Requirements of Specimens for
Microbiological Tests
.

Transport Condition Specimen

Body fluids Bone
Inner ear Corneal scrapping
Immediately at room
Foreign bodies Gastric aspirate
temperature
Suprapubic aspirate of urine
Prostatic sample
 Transport Requirements of Specimens for
Microbiological Tests
.
Transport Condition Specimen
W/in 15 minutes at room temperature CSF
Within 2 hours at room temperature Blood or bone marrow
Immediately at 4 C
̊ Gastric Biopsy
Indwelling/straight
Within 2 hours at 4 ̊C
catheter (Urine sample)
 Transport Requirements of Specimens for
Microbiological Tests
.
Transport Condition Specimen
Abscess
Outer Ear
Within 24 hours at room Conjunctiva Specimen
temperature Genital tract specimen (JEMBEC)
Hair, Nail, Skin Scrapings
Respiratory and Tissue specimen
 Transport Requirements of Specimens for
Microbiological Tests
.

Transport Condition Specimen

Rectal swab
Within 24 hours at 4 ̊C Stool culture
Clean voided midstream urine
Specimens Prioritization:
Level Description Specimen
.
1 Amniotic fluid, blood, brain, CSF,
Critical/Invasive heart valves, pericardial fluid
2 Bones, feces, sputum, tissue, other
Unpreserved body fluids not listed under level 1
3 Quantitation required Catheter tip, urine, tissue

4 Urine, feces, and swab in holding

Preserved media
Specimens Prioritization:
NOTE:
▪ . When specimen is received with multiple requests but the amount of
specimen is insufficient, the clinician should inform the laboratory
staff which of the tests should be prioritized.
▪ When multiple specimens arrive at the same time, priority should be
given to those that are most critical such as CSF, tissue, blood and
sterile fluids.
▪ Urine, throat, sputa, stool or wound drainage are specimens that can be
stored and processed later.
▪ AFB, viral, fungal specimens can be processed by batch.
Specimens Rejection:
1. The information on the label does not match the information on the
requisition
.
slip.
2. The specimen is transported at an improper temperature.
3. The transported specimen has not been placed in the proper medium.
4. The quantity of specimen is not sufficient (QNS) for testing.
5. The transport of specimen exceeds the two hours’ post-collection period.
6. The specimen is preserved in a fixative (formalin).
7. The specimen has been reserved for anaerobic culture from a site known
to have aerobes as part of the indigenous microbiota (vagina and mouth).
8. The specimen is leaking.
Specimens Rejection:
9. The. specimen has already dried up upon transporting to the laboratory.
10. The specimen being processed has a questionable medical value.
11. More than one specimen from the same source (except for blood) and from
the same patient is submitted on the same day,
12. A single swab is submitted with multiple request for various organism.
13.Expectorated sputum in which the gram stain reveals <25 WBC
and > 10 Epithelial Cells.
 Critical Panic Results in a Clinical Microbiology

1. Positive
.
CSF Gram stain and culture
2. Gram stain suggestive of C. perfringens (gas gangrene)
3. Positive AFB stain
4. Positive blood culture
5. Presence of Streptococcus pyogenes in a surgical wound
6. Positive antibiotic-resistant bacteria
7. Positive for Legionella, Francisella, and Brucella
Gram Stain
▪ It is the most commonly used differential stain in the clinical
microbiology laboratory.
▪ Reagent: Crystal Violet, Iodine, Acetone-alcohol, Safranin
▪ Principle:
Bacteria with thick cell wall containing teichoic acid retain the
crystal violet-iodine complex dye after decolorization and
appear purple, which mean that they are Gram (+). Other
bacteria with thinner cell walls contain lipopolysaccharides do
not retain the dye complex and appear deep pink or red, which
means that they are Gram (-).
Gram Stain
Gram- Gram-
positive negative
bacteria bacteria
Primary Stain
Mordant
Decolorization
Secondary Stain
GRAM-STAIN
Gram Stain Reaction

Bacillus Staphylococcus Escherichia

Mycobacteria Streptococcus Klebsiella
Clostridium Micrococcus
C. diphtheriae Salmonella
Listeria Shigella
Nocardia, Proteus
Streptomyces Neisseria Pseudomonas
Moraxella Vibrio
▪ A CULTURE AND SENSITIVITY
(C & S) TEST is the primary
procedure performed in
microbiology.
▪ It is used to detect and
identify microorganisms
and to determine the most
effective antibiotic
therapy.
▪ Results are available within
2 days for most bacteria.
BACTERIOLOGY SECTION
 Tests Performed in the Microbiology Section
Detects acid-fast bacteria, culture
Acid-fast bacillus (AFB)
including Mycobacterium tuberculosis
Blood culture Detects bacteria and fungi in the blood
Detects the presence of and determines the
Fungal culture
type of fungi
Detects the presence of and aids in the
Gram stain
identification of bacteria
KOH direct mount For examination of fungal elements
technique (Hypha, Conidia, etc.).
Detects nonvisible blood (performed on
Occult blood
stool samples)
Detects parasitic infection (performed on
Ova and Parasites (O & P)
stool samples)