MLSP 112 INTRO TO BACTERIOLOGY

Prepared and organized by: Jericho Morales

BACTERIOLOGY, MYCOLOGY BACTERIA

AND VIROLOGY - Prokaryotic unicellular organisms that
lack a true nucleus and nuclear membrane
- Single, closed, circular chromosomes of
double-stranded DNA called NUCLEOID.
- PLASMIDS: small circular molecules of
extrachromosomal circular DNA
- May either be GRAM POSITIVE or
GRAM NEGATIVE
- BINARY FISSION: asexual reproduction
of bacteria
- Bacteria size: range from 0.2 to 2μm in
diameter and 1 to 6μm in length

Biological Safety Cabinet GRAM STAIN

CLASS of Function/s - Principal

Biosafety stain used for
Cabinet microscopic
Class I -Allow room air to pass into examination of
cabinet and around bacteria
the area and material within, - First devised
sterilizing only the air by Hans
to be exhausted Christian Gram
-consist of negative pressure during the late
-operated in open front 19 th century
- Divide most bacterial species into two
Class II -air flows in “sheets” , which large groups:
Class IIA serves as a barrier to 1. Gram-positive: take up the basic dye,
Class IIB particle from outside the crystal violet
cabinet 2. Gram-negative: allow crystal violet dye
-Direct the flow of to wash out easily with decolorizer alcohol
contaminated air into the or acetone
filters
-also called as VERTICAL
LAMINAR FLOW BSCs
-self-contained, and 70% of
the air is recirculated.
-selected for radioisotopes,
toxic chemicals or
carcinogenic samples

Differences between Gram Positive

and Gram Negative

1 PROPERTY OF ECHO
 MLSP 112 INTRO TO BACTERIOLOGY
Prepared and organized by: Jericho Morales
Four basic morphological types of
Collection and handling of
GRAM GRAM Clinical Specimen
POSITIVE NEGATIVE
Collection
CELL WALL Thick Thin 1. Safety
(Peptidoglycan) (peptidoglycan) - UNIVERSAL PRECAUTIONS
are followed throughout
LIPOPOLYSACCHARIDE Absence Presence the collection and handling
process.
GRAM STAINING Purple Red or Pink - Persons collecting or
handling specimens should
REPRESENTATION Picture of Picture of gram wear gloves and a laboratory
Gram positive negative gown
“PURPLE or “PINK” - Eye protection should also
VIOLET” Result be worn if splashing
Result - Accidents or injuries
bacteria (needle prick) must be reported
immediately.
1. COCCI: spherical-shaped cells
2. BACILLI: rod-shaped cells 2. General Guidelines
3. SPIRILLA: spiral shaped cells - Specimen should be from the infection
4. VIBRIOS: comma- shaped cells site and not
contaminated by the surrounding area
- Specimen should be collected before
antimicrobials are
administered (Antibiotics)
- Appropriate collection devices and
containers should be used
and must be STERILE! ASEPTIC
TECHNIQUE is required.
- Specimen container should LABELED with
patient’s identification, the date and time of
collection and the source of
specimen

Collection form various body sites

I. Throat

Two types of bacterial metabolism

1. FERMENTATION
- Process by which bacteria catabolize
carbohydrates to produce energy

2. RESPIRATION
- also known as oxidation
- Process of bacterial energy generation
rather than fermentaion - Tongue should be depressed before
swabbing between the tonsillar pillars and
2 PROPERTY OF ECHO
 MLSP 112 INTRO TO BACTERIOLOGY
Prepared and organized by: Jericho Morales
behind the uvula.
- The cheek, tongue, and teeth should NOT
be touched.
II. Sputum
- Expectorated specimens from deep cough
should be collected into
a sterile specimen cup
-NOTE: Early morning specimens are the
best.
IV. Stool
- Should never be taken from the toilet and
should not be contaminated with urine
V. Urine
- Midstream clean-catch is the most
common collection method.
- Culture for catheterized urine specimens
usually have less contaminating bacterial
flora
VI. Blood
- 2 to 3 cultures should be collected at
random times during 24- hour period.
- Skin is disinfected with 70% alcohol,
followed by Iodine
- ADULT: 20-30mL of blood per culture is
collected
- INFANTS: 1-5mL of blood per culture is
collected
VII. Cerebrospinal fluid
- Should be collected aseptically by
PHYSICIAN
- Should be processed immediately and not
exposed to heat or refrigeration
VIII. Genital Tract
a. MEN (penile discharge)
- Exudates may be expressed from the
urethral orifice or a small-diameter swab
inserted 3 to 4 cm into the urethra.
- WOMEN (vaginal discharge)-
- A sterile swab is inserted into the cervix
with an aid of speculum
- Swab is rotated and allowed to remain for
a few seconds
Handling &Transport
1. Anaerobic specimens must be
transported in an anaerobic system.
2. Swab samples are not allowed to dry out
3. Most specimen can be held at 2ºC to 8ºC
if transport cannot occur immediately
EXCEPT:
a. Temperature-sensitive organisms
b. Blood culture bottles
c. CSF (Cerebrospinal fluid)
PROCESSING
I. Media Selection
1. NUTRITIVE MEDIA
- Support the growth of a wide range of
microorganisms and are considered non
selective because, the growth of most
organisms is supported
- Example: Blood Agar Plate and Chocolate
Agar
2. DIFFERENTIAL MEDIA
- Microorganisms that can be distinguished
on the basis of certain growth
characteristics evident on the medium
- Example: Blood agar plate
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 MLSP 112 INTRO TO BACTERIOLOGY
Prepared and organized by: Jericho Morales
2. Specimen contaminated with barium or
3. SELECTIVE MEDIA other foreign substances
- Support the growth of one group of 3. 24- hour urine or sputum collections
organisms, but not another, by adding 4. Saliva instead of sputum
antimicrobials, dyes, or alcohol to a 5. Unrefrigerated urine specimens 2 hours
particular medium. or more post- collection
- Example: MacConkey Agar, Columbia
agar with colistin and nalidixic acid (CNA)

4. BACKUP BROTH
- Also called supplemental or enrichment
broth
- For detection of small numbers of
organisms present
- Example: Thioglycollate (Thio) broth,
brain-heart infusion broth (BHIB) and
tryptic soy broth (TSB)

5. SPECIALIZED MEDIA
- Used when specific organisms are
suspected
- Example: Mannitol salt agar, bismuth
sulfite agar, Campylobacter agar,
Thiosulfate-citrate-bile salts- sucrose
(TCBS) agar

Incubation
1. 35ºC-37ºC: Normal incubation
temperature for bacterial cultures
2. Anaerobic jars, bags, or an anaerobic
chamber are appropriate for incubation of
anaerobic cultures
3. 42ºC to 45ºC: for stool cultures for
detection of Campylobacter jejuni

Specimen Types to be rejected

1. Specimen receive d in nonsterile or
contaminated containers
4 PROPERTY OF ECHO