BACTERIOLOGY MLS – 110

LECTURE 5 - 6
CULTIVATION OF BACTERIA 2. Solid
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– • With solidifying or hardening agent
TERMINOLOGIES • contains Agar (1.5 – 3%)
1. CULTURE ➢ Doesn’t contain nutritional value
• (noun) Growth of microorganisms ➢ Come from “red algae” / seaweeds (Kingdom
• (verb) To cultivate, isolate, grow, plant an organism Plantae, Phylum Rhadophyta)
2. INOCULATE/PLANT/CULTIVATE • Examples:
• Introduction of microorganisms in a culture media ➢ Nutrient Agar (NA)
3. TRANSPLANT/SUBCULTURE ➢ Chocolate Agar Plate (CAP)
• The transfer of microorganisms from one culture ➢ Blood Agar Plate (BAP)
media to another culture media. 3. Semi-Solid
4. FASTIDIOUS ORGANISM • Contains less amount of agar (0.5 – 1%)
• Any organism which has very complicated nutritional • For studying motility of bacteria
requirements, meaning it will not grow without • Examples:
specific factors present or in specific conditions. ➢ Sulfide Indole Motility (SIM)
• Examples: Neisseria gonorrhoeae, Campylobacter, ➢ Motility Indole Ornithine (MIO)
Helicobacter, Mycobacterium 4. Bi-Phase
5. NON-FASTIDOUS ORGANISM • Liquid or solid component of media used to isolate
• Do not require any special nutrition supplement or • Example:
conditions to grow. ➢ Casteneda medium (Brucella spp.)
• Examples: E. coli, S. aureus, P. aeruginosa
6. INDICATORS II. According to Composition
• Added to the medium to visibly indicate the defining 1. Base / Basal / Simple
characteristics of a microorganism. • Basic requirements for non-fastidious organism to
7. INHIBITORS grow
• Chemical substances can be added to culture 2. Synthetic
media to inhibit certain bacteria. • Chemically defined medium
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– • Exact chemical composition is known to users
TYPES OF CULTURE • Examples: Difco Oxoid
1. Pure Culture 3. Non-Synthetic
• Type of culture where a single species of • Complex media
microorganism growing in a culture medium - • Exact chemical composition is not known to user
Example: Escherichia coli 4. Tissue Culture
2. Mixed Culture • Medium that contains living tissue cells used in
• There are two or more wanted organisms / species / isolating microorganisms that cannot be drawn on
genera growing in a culture media - Example: artificial media.
Klebsiella pneumoniae with Escherichia coli • Examples:
3. Contaminated Culture ➢ McCoy cells - Isolate Chlamydia spp. (obligate
• Accidentally unwanted organisms growing on a intraparasites)
culture medium ➢ Chick Embryo - Isolate Rickettsia spp.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
III. According to Manner of Dispensing/Formation
SOURCES OF CONTAMINATION
1. PLATED
✓ Environment
• usually contained in a container that can be made
✓ Materials Used for Transfer of Specimen to Culture
of glass (pyrex) or disposable plastic. This container is
Media
commonly known as PETRI DISH after its proponent
✓ Technologies Performing the Procedure
Richard Julius Petri.
✓ Culture Media Itself
A. Types of plated medium: (Made up of plastic or glass)
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
❑ Small
CLASSIFICATION OF CULTURE MEDIA
o size of a standard saucer plate;
I. According to Physical State
o Diameter: 60 mm; Thickness: 15 mm - 60mm/15mm
1. Liquid
❑ Large/Big
• Broth medium - Doesn’t contain solidifying or
o size of a standard dish plate (Usually used in AST)
hardening agent
o Diameter: 150 mm; Thickness: 15 mm -
• Examples:
150mm/15mm
➢ Nutrient Broth (NB)
B. Storage of Plated medium
➢ Trypticase Soy Broth (TSB)
❑ Refrigerator - Upside-down orientation
➢ Brain Heart Infusion Broth (BHIB)
❑ Incubator - Upside-down orientation
➢ Mueller Hinton Broth (MHB)
❑ Shelf life - One week
➢ Thioglycolate Broth
C. General Procedure
➢ Selenite F
❑ Weigh
❑ Dissolve
❑ Homogenize

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 BACTERIOLOGY
LECTURE 5 - 6
❑ Sterilize
❑ Dispense - Before 40 ˚C (before it solidify)
D. Heat labile
❑ Has temperature requirements; has ingredients or
components that are destroyed when exposed to
high temperature
❑ Do not use autoclave to sterilize, instead by filtration,
boiling, and other means of sterilization (inspissations,
tyndallization)
❑ Examples:
➢ Salmonella Shigella Agar (SSA)
➢ Thiosulfate Citrate Bile salt Sucrose Agar (TCBS)
➢ Lowenstein Jensen (LJ) Selenite-F Christiansen’s
Urea Broth
2. Tubed
• Usually container in glass tubes such as Wassermann
tubes with different volume capacity (3mL, 5mL,
10mL) or in a tube with a flat bottom and a screw
cap. There are three (3) common types:
A. Types of Tubed medium
❑ Liquid/ Broth
❑ Solid
❑ Butt/Deep
❑ Slant - Example: Simmon Citrate Agar
❑ Butt / Slant - Examples: Lysine Iron Agar, Triple
Sugar Iron Agar
B. General Procedure in Tubed media preparation
❑ Weigh
➢ Weigh the powder-formed bacteria / culture media
(commercially prepared) on a foil using analytical
balance - Expressed in grams (mass)
❑ Dissolve
➢ Measure volume of the solvent using graduated
cylinder
➢ Required solvent is distilled water (1 L / 1 000 mL)
➢ Example: Triple Sugar Iron Agar = 32 g Distilled
water = 1000 mL Before transferring the distilled
water onto the Erlenmeyer flask, divide it into
two; then dissolve the powder and mix; after
mixing, pour the remaining distilled water.
❑ Homogenized / Homogenization
➢ Completely dissolving by boiling using hot plate
until the solution is clear
❑ Dispense
➢ Dispense in each test tube
❑ Sterilize
➢ Sterilize using autoclave
➢ Pressure: 15 psi
➢ Temperature: 121 ˚C
➢ Time: 15 – 20 mins (dependent on the
components of autoclave)
➢ Moist: higher pressure in the autoclave
3. Bottled
• culture media contained in a glass bottle that is
usually used for blood culture
Types of Bottled Culture Media
❑ CASTANEDA
➢ a biphasic medium that is obsolete already that
was used for blood culture to isolate Brucella
abortus.
RDME | BSMT – 3C
MLS – 110
❑ SEPTI CHEK ®
➢ a broth slide system which is an innovation of the
Castaneda bottle which consists of slide paddles
containing Chocolate Agar, MacConkey, and
Malt Extract Agar (selective for yeasts and fungi)
attached to the top of a standard broth bottle.
This is a manual blood culture system which
requires daily inversion or at least twice weekly
to bathe the slide paddle with the broth culture
medium, thereby allowing infrequent
subcultures, hence less contamination.
❑ BACTEC ®
➢ first automated growth detection system for
blood cultures which detects radiolabeled
carbon (14C). In the bottle, there is a 14C-
labelled substrate which is then utilized or
degraded by bacteria which liberates CO2
which is then measured in an ionization
chamber. The amount of CO2 generated, if
exceeds the threshold set, will be detected as a
positive growth.
IV. According to Function/Uses
1. General Purpose Culture Media
• Type of culture media use to grow non-fastidious
organism
• Use as a base medium in preparing complex media
for fastidious organism
• Examples:
➢ Nutrient Agar
➢ Nutrient Broth Trypticase Soy Broth
➢ Trypticase Soy Agar
➢ Brain Heart Infusion Broth
➢ Brain Heart Infusion Agar
2. Enriched Culture Media
• Media that contains enriching substances/additives
to enhance the growth of fastidious organism
A. Sources of additives
• 5% defibrinated sheep’s blood (best source)
• Horse blood
• Rabbit’s blood
• Human blood (least because it may contain
antibodies that may inhibit the growth of bacteria)
• Example:
➢ To prepare Blood Agar Plate (BAP):
➢ Before it solidify, put blood on it
➢ It turns Color red because of the blood
➢ Example:
➢ To prepare Chocolate Agar Plate:
➢ After putting the blood, put it on the hot plate
➢ Hemoglobin component of blood burst and will
cause the brown color
3. Enrichment Culture Media
• Used to increase pathogens in a specimen wherein
pathogenic are outnumbered by nonpathogens
• Examples:
➢ Selenite-F – For Salmonella and Shigella (stool
sample)
➢ Alkaline Peptone Water - For Vibrio spp.
➢ Thioglycolate - For sputum sample; anaerobe
and aerobe
2
 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
4. Selective Culture Media • Example: Streptococcus pneumoniae
• Contains inhibitory substances or any chemicals c.) Gamma hemolytic
used to inhibit growth of unwanted organisms and • There’s no hemolysis; characterized by red color
have favor the desired ones around the colonies
A. Inhibitors • Example: Streptococcus bovis
❑ Gram positive inhibitors (*Gram negative will grow)
❑ Examples: 2. Mannitol Salt Agar (MSA)
➢ Bile salts • Differential and selective culture medium
➢ Citrate a.) Selective
➢ Antibiotics ❑ Inhibitory substance: High salt concentration (grows
➢ Dye halophilic / highly selective on halophilic
✓ Crystal Violet ❑ Example: Staphylococcus spp.
✓ Gemtian Violet b.) Differential
✓ Malachite Green ❑ Indicator: phenol red (pH indicator) – color pink / red
✓ Eosin ❑ Source of carbohydrates / energy is: mannitol or
✓ Methylene Blue glucose
❑ Gram negative inhibitors (*Gram positive will grow) ❑ Mannitol Fermenter - Yellow colonies
❑ Examples: ❑ Example:
➢ Potassium Tellorite ➢ Staphylococcus aureus
➢ Sodium Azide ❑ Non-Mannitol Fermenter - Red / pink / fuchsia pink
➢ Alcohol colonies
➢ Antibiotics ❑ Examples:
B. Swarming phenomenon ➢ Staphylococcus epidermidis
❑ Wave like growth of colonies growing in a solid ➢ Staphylococcus saprophyticus
culture medium ➢ Staphylococcus hyicus
➢ Example organism: Proteus spp. ➢ Staphylococcus lugdunensis.
❑ To prevent swarming:
➢ Choral Hydrate 3. MacConkey Agar (MAC)
➢ Alcohol • Differential and selective culture medium
➢ Trimethoprim lactate • selective for gram negative bacilli
a.) Selective (inhibitor)
5. Differential Culture Media ❑ Crystal violet
• Contains indicators to differentiate group of bacteria ❑ Bile salt
• Used in grouping bacteria b.) Differential
• Examples of indicators: ❑ Indicator: neutral red (pH indicator) – around 7 pH
➢ pH indicator ❑ Source of carbohydrates / energy: LACTOSE
➢ Hydrogen Sulfide (H2S) (ferric/sulfur) ❑ May or may not use lactose as source of energy
➢ Blood ❑ Degrade lactose into glucose and galactose
(beta galactosidase); may produce acid.
❑ Lactose Fermenter
➢ Pink colonies
➢ Rapid Lactose Fermenter
➢ Have beta galactosidase and lactose
permease
❑ Late Lactose Fermenter
➢ light pink
➢ Have beta galactosidase but, no lactose
permease
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ❑ Non-Lactose Fermenter
Examples of Selective and Differential Culture Media ➢ Colorless colonies
1. Blood Agar Plate (BAP) ➢ Will not produce acid
• indicator is blood
• *universal but differential agar 4. Salmonella Shigella Agar (SSA)
Three Groups of Bacteria based on their colony appearance • Differential and selective culture medium
in BAP • Same as MacConkey Agar, but superior on it
a.) Beta hemolytic • Pink
• Group of bacteria that exhibit complete hemolysis; a.) Selective
characterized by “clear- zone” around the colonies ❑ Brilliant green
• Example: Streptococcus pyogenes ❑ Bile salt
b.) Alpha hemolytic b.) Differential
• Type of bacteria that exhibit incomplete hemolysis; ❑ Indicator:
characterized by greenish discoloration around the ➢ neutral red (pH indicator) – around 7 pH
colonies

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 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
➢ H2S (Ferric Ammonium Citrate with Sodium b.) Differential
Thiosulfate); causes the blackening, black ❑ Indicator:
precipitate at the center ➢ Eosin Y, Methylene Blue (pH indicator)
➢ Source of carbohydrates / energy: LACTOSE ➢ around 7 pH
o May or may not use lactose as source of ➢ Source of carbohydrates / energy: LACTOSE
energy ➢ May or may not use lactose as source of energy
o Degrade lactose into glucose and ➢ Degrade lactose into glucose and galactose
galactose (beta galactosidase); may (beta galactosidase); may produce acid.
produce acid ❑ Lactose Fermenter
❑ Lactose Fermenter ➢ Pink colonies
➢ Pink colonies ➢ Rapid Lactose Fermenter
➢ Rapid Lactose Fermenter ➢ Have beta galactosidase and lactose
➢ Have beta galactosidase and lactose permease
permease ➢ Pink except: Escherichia coli (greenish metallic
➢ H2S (+) - Pink with black center; H2S (-) - sheen)
Pink ❑ Late Lactose Fermenter
❑ Late Lactose Fermenter ➢ Have beta galactosidase but, no lactose
➢ Have beta galactosidase but, no lactose permease
permease ❑ Non-Lactose Fermenter
➢ H2S (+) – light Pink with black center; H2S (-) - ➢ Colorless colonies, sometimes dark purple / dark
light Pink blue
❑ Non-Lactose Fermenter
➢ Colorless colonies
➢ H2S (+) - With black center; H2S (-) - Colorless

5. Hektoen Enteric Agar (HEA)

• Differential and selective culture medium
• Green
a.) Selective
❑ Brilliant Green
❑ Bile Salt
b.) Differential
❑ Indicator:
➢ BROMTHYMOL BLUE (pH indicator)
➢ H2S (Ferric Ammonium Citrate with Sodium
Thiosulfate); causes the blackening, black
precipitate at the center 7. Thiosulfate Citrate Bile Salt Sucrose (TCBS)
➢ Source of carbohydrates / energy: LACTOSE • Differential and selective culture medium
➢ May or may not use lactose as source of energy • Green - Heat labile
➢ Degrade lactose into glucose and galactose • Grows gram negative - Highly selective in Vibrio spp.
(beta galactosidase); may produce acid a.) Selective
❑ Lactose Fermenter ❑ Methylene Blue
➢ Yellow / Orange colonies ❑ Eosin
➢ Rapid Lactose Fermenter b.) Differential
➢ Have beta galactosidase and lactose ❑ Indicator: bromthymol blue (pH indicator)
permease ➢ alkaline
➢ H2S (+) - Yellow / Orange with black center; H2S ➢ source of carbohydrates: SUCROSE
(-) - Yellow / Orange ➢ Sucrose Fermenter - Yellow / Orange colonies
❑ Late Lactose Fermenter o Example: Vibrio cholerae
➢ Have beta galactosidase but, no lactose ➢ Non-Sucrose Fermenter - Green / Blue colonies
permease H2S (+) - Yellow / Orange with black o Example: Vibrio parahaemolyticus
center; H2S (-) - Yellow / Orange
❑ Non-Lactose Fermenter 8. Lowenstein Jensen (LJ)
➢ Green / Blue colonies • Selective culture medium
➢ H2S (+) - Green / Blue with black center; H2S (-) - • Green
Green / Blue • Heat labile
• Highly selective in Mycobacterium spp.
6. Eosin Methylene Blue (EMB) a.) Selective
• Differential and selective culture medium ❑ Malachite Green
a.) Selective ❑ High concentration of protein
❑ Methylene Blue
❑ Eosin

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 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
9. Bismuth Sulfite Agar (BSA) Additives/Anticoagulants
• Selective culture medium 1) 0.025-0.030% SPS (Sodium Polynaethol sulfonate)
• Highly selective in Salmonella spp. • Higher concentration of SPS can inhibit the following:
• Source of carbohydrates / energy: GLUCOSE ➢ Gardnerella vaginalis
• Examples of Selective Culture Media: selective agar ➢ Neisseria gonorrhea
for Neisseria spp. ➢ Streptobacillus monoliformis
➢ Peptostreptococcus anaerobius
• to counteract: add 1% of Gelatin
2) Heparin
• Often used for viral cultures and isolation of
Mycobacterium spp.
• may inhibit growth of gram (+) bacteria ang yeast.
• Citrate, EDTA, or other anticoagulants – Not used in
microbiology because their efficacy has not been
demonstrated for a majority of organisms.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
10. Special Culture Media Transport Medium
• Use to grow specific type of microorganism 1. Amie’s Medium - Has gel inside to preserve and to
• Examples: prevent drying of specimen; most type of specimen
➢ Mannitol Salt Agar (MSA) - For Staphylococcus especillay respiratory samples.
spp. 2. Cary-Blair Medium - Has fluid inside to preserve and
➢ Thiosulfate Citrate Bile salt Sucrose Agar (TCBS) - to prevent drying of specimen; STOOL spx.
For Vibrio spp. 3. Stuart’s Medium - Has gel or charcoal to preserve and
➢ Bismuth Sulfite Agar (BSA) - For Salmonella spp. to prevent drying of specimen; some types of spx and
➢ Lowenstein Jensen (LJ) - For Mycobacterium for Neisseria gonorrhoeae.
spp. 4. Transgrow Medium - Recommended transport
➢ Loeffler’s Coagulated Serum - For medium for Neisseria gonorrhoeae - Has Chocolate
Corynebacterium spp. Agar inside with inhibitory substances (Colistin,
Vancomycin, and Nystatin)
11. Transport Culture Media –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
• Culture media use to maintain the viability of Unacceptable Specimens
microorganisms when there is an anticipated delay 1. Anaerobic Set ups - Sputum, stool, and urine (except
in bringing the specimen from patient to laboratory suprapubic aspiration)
or to other location. 2. Samples with Fixatives - Fixatives are formalin,
• Has charcoal component to maintain moisture alcohol, acetone, etc
• Examples: 3. Incorrect Label of Specimen
➢ Amies 4. Samples Transported at the Improper Temperature
➢ Stuart 5. Quantity Not Sufficient (QNS) Samples
➢ Transgrow 6. Specimen is Leaking
➢ Cary-Blair 7. Exceeds 2 hrs Collection and Not Preserved
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 8. Specimen Dried up
COLLECTIONS AND PROCEDURES OF MICROBIAL SPECIMEN 9. Foley Catheter Tip (Prone to Contamination)
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Function of Microbiology Specimen for Microbial Collection
• To isolate, identify microorganism for investigation of I. BLOOD
infection 1. Indications of Blood Testing/Culture
• To test for antimicrobial for the treatment of patients • Fever of unknown origin
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– • Bacteremia - Merely presence of bacteria in the
Two Methods of Preserving Bacterial Specimens blood stream without multiplication
1. Physical Method • Septicemia - Merely presence of bacteria in the
• Refrigerator - Storage in 4 ˚C for urine, stool, swabs, blood stream with multiplication
sputum, catheters • Typhoid fever - Causative agent (Salmonella spp.)
• Storage in 35 – 37 ˚C for Cerebrospinal Fluid (CSF), may be isolated from blood, stool
blood cultures • Endocarditis - Infection of the endocardium, which is
• Incubator - 35 – 37 ˚C the inner lining of your heart chambers and heart
2. Chemical Method valves. (HACEK organisms)
• Should refrain from doing for it may harm • Brucellosis - Caused by ingestion of unpasteurized
microorganisms milk or undercooked meat from infected animals
• Boric Acid - Preservation for urine • Only clinical specimen used to isolate the causative
• Phosphate Buffer Saline (PBS) - Preservation for stool agent is blood
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

RDME | BSMT – 3C 5
 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
• Leptospirosis - Caused by corkscrew shaped • Thick pellicle forms on the surface of the medium with
bacteria called Leptospira spp. - Use blood to hemolysis - Bacillus spp, saprophytic fungi
isolate • Marked hemolysis with unpleasant odor and gas
2. Collected through Venipuncture under pressure - Clostridium spp.
• Antiseptics • Less gas but with foul odor - Bacteroides spp.
a) Iodine / Iodophor • No change in the medium only by subculturing on
b) 70 % alcohol chocolate agar - Haemophilus spp. (smallest
• Two Set ups bacteria)
a) Aerobic Culture
b) Anaerobic Culture II. URINE
3. Blood Anticoagulant/Additive • Collected in a wide mouth, sterile, screwcap
• 0.025 – 0.03 % Sodium Polyanethol Sulfonate (SPS) container
• Characteristics 1. Ways of Collection
❑ Prevents phagocytosis ❑ Midstream clean catch method
❑ Prevent complement activation ➢ Most convenient
❑ Neutralizes some antibiotics ➢ Wash the genitalia, void the first output of urine
• Neutralizers ➢ Ideal volume is 100 – 150 mL
❑ Penicillinase - Neutralize the effect of penicillin ❑ Suprapubic aspiration
❑ Para-Amino Benzoic Acid (PABA) - Neutralize ➢ Collected by skilled physician, using a syringe
sulfonamides ➢ Can be aerobe or anaerobe - Most sterile
❑ Magnesium sulphate - Neutralize tetracycline ❑ Catheterization - Make use of Foley catheter (rubber
4. Ideal Volume tube)
• Higher the volume of blood, the higher the rate of ➢ Collected by skilled physician
isolating bacteria ➢ Prone to contamination
• Ratio of blood culture medium = 1:10 - 10 mL blood: ➢ Not intended for urine collection
100 mL culture medium ➢ For patients who are having a difficulty in
• Amount of blood for infants = 5 – 10 mL urinating
• Amount of blood for adults = 10 – 20 mL ➢ Types of Catheterization:
5. Blood Culture Medium a) Urethral - Tube is put directly onto urinary
• Aerobic / Anaerobic bladder
❑ Brain Heart Infusion Broth (BHIB) b) Ureteral - Tube is put all throughout the
❑ Thioglycolate ureter from the genitalia
❑ Trypticase Soy Broth ❑ Plastic bag
❑ Nutrient Broth ➢ “Wee bag”
• Brucella Broth / Wisconsin Medium / Castañeda ➢ Intended for pediatric patients
Medium ➢ Placed on genitalia of infants
• Fletcher’s Medium / Ellinghausen-McCullough- 2. Infections
JohnsonHarris ❑ Cytitis - Infection of urinary bladder
❑ Pyuria - Presence of puss cells in urine
❑ Dysuria - Painful urination

3. Microbiology Tests
❑ Gram Stain
❑ Culture and Sensitivity
a) Culture Media
➢ MacConkey Agar (MAC) - For gram negative
➢ Blood Agar Plate (BAP) - As general media (for
gram positive and negative)
➢ Phenylethyl Alcohol Agar (PEA) - For gram
positive
6. Signs of Growth in Blood Culture b) Storage - 35 ˚C for 12-48 hours only
• Uniformly turbid medium with gas bubbles - Gram c) Counting Colonies
negative rods ➢ Use of calibrated loop for plating = 0.01 mL or
• Less distinct turbidity with greenish tint - 0.001 mL / 1uL
Streptococcus pneumoniae, Neisseria meningitidis ➢ One colony = 1 000 CFU/mL
• Cotton ball colonies on top of sedimented red cell ➢ > 100 000 CFU/mL = Urinary Tract Infection (UTI)
with clear upper layer of broth - Non-hemolytic ➢ < 100 000 CFU/mL = if with 3 or more species,
Streptococcus spp. insignificant
• Turbid with marked hemolysis of blood - Beta ➢ Estimation:
hemolytic Streptococcus spp. o Line of inoculation = +/- 5 000 colonies
• Large jelly-like coagulum throughout the broth - o Streak = 10 000 colonies
Staphylococcus aureus –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

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 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
Organisms Most Frequently causing Urinary tract Infection b.) Mycobacterial Culture
(UTI) ➢ Culture medium:
• Lowenstein Jensen (LJ)
c.) Fungal Culture
➢ Culture medium:
• Sabouraud’s Dextrose Agar (SDA)
d.) Viral Culture:
➢ Cell / Tissue Culture

IV. CEREBROSPINAL FLUID (CSF)

III. SPUTUM
• Normal fluid used as lubricant
• Fluid that is a product of either inflammatory or
• Source of nutrient for the nervous system
infectious condition
• Indication of meningitis / meningococcemia
• Formed in the lower respiratory tract (lungs)
• Collected by skilled physician, neurosurgeons; using
• Can be used to diagnose pneumonia
syringe
1. Possible Indications • Recommended volume is 8 – 12 mL divided onto 3
❑ Lower respiratory tract infection separate containers
❑ Bacterial pneumonia • Clear color
❑ Pulmonary tuberculosis
1. Ways of Collection
2. Way of Collection
❑ Lumbar puncture
❑ Expectoration / deep coughing – Expectorated early
➢ Routinely performed
in the morning (most concentrated)
➢ Patient is instructed to lay down, lower
3. Microbiology Tests
extremities are bended
❑ Microscopic Examination
➢ Collected in the third & fourth or fourth & fifth
a.) Gram stain
lumbar area
➢ For identification of bacterial pneumonia
❑ Cisternal Tap
o Pus cells - More than 25/LPF - Increase is
➢ For babies
significant
➢ Ventricular Tap
o Epithelial cells - Less than 10/LPF - Increase
2. Three Sterile Containers (Tube)
means contamination
❑ Tube 1- Hematology (for cell count and differential
o Pulmonary Alveolar Macrophages -
count) or non-routine studies
Indicates the sputum in the specimen
❑ Tube 2 - Serology and Clinical Chemistry
➢ To validate that specimen came from lower
➢ Examination of electrophoresis and ELISA testing for
respiratory tract
serology
b.) Acid Fast Bacilli stain
➢ Sugar, protein, lactose dehydrogenase for clinical
➢ For pulmonary tuberculosis screening
chemistry
➢ Needs 3 samples from tuberculosis patients to
❑ Tube 3 - Microbiology
increase the yield of positive results
➢ Most sterile
➢ Use of Auramine-Rhodamine dye
➢ Centrifuge in 15 mins for 3 000 rpm
o Direct smear
3. Microbiology Tests
o Concentration method - To increase
a.) Microscopic
sensitivity of procedure using digestive or
❑ Gram stain
decontaminating agent
❑ Acid Fast Bacilli stain
❑ Culture Examination
❑ India ink method
a.) Bacterial Culture
b.) Cultural
➢ Bacterial pneumonia
❑ Bacterial culture
➢ Different Culture Media
➢ Thioglycolate
o Blood Agar Plate (BAP) or Nutrient Agar (NA)
➢ Chocolate Agar Plate (CAP)
✓ General Medium
❑ Mycobacterial culture
✓ Incubated in incubator for 35 – 37 ˚C
➢ Lowenstein Jensen (LJ) medium
o MacConkey Agar (MAC) or Eosin Methylene
➢ Fungal culture
Blue (EMB)
➢ Sabouraud Dextrose Agar (SDA)
✓ Selective and Differential Medium
❑ Viral culture
✓ Incubated in incubator for 35 – 37 ˚C
➢ Cell / tissue culture
o Chocolate Agar Plate (CAP)
❑ Hematology
✓ Enriched Medium
➢ Cell count
✓ Incubated in capnophilic environment
✓ White Blood Cell count
(candle jar)
✓ White Blood Cell differential count
o Thioglycolate
✓ Enrichment Medium
✓ Incubated in capnophilic environment
(candle jar)

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 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
V. STOOL • Found in different body cavities in low quantities / in
• For diagnosis of gastroenteritis / diarrhea large quantities (effusion): Transudates (product of
• Considered as non-sterile mechanical processes) and Exudates (product of
• Collection in a wide mouth, sterile, screw type inflammatory / infectious agents)
container 1. Fluid
1. Culture ❑ Synovial fluid - Lubricant in joints
a) General ❑ Amniotic fluid - Normal fluid present in pregnant
❑ Blood Agar Plate (BAP) women
❑ MacConkey Agar (MAC) ❑ Serous fluid
❑ Salmonella Shigella Agar (SSA) a.) Pleural fluid - Found between pleural sac and
❑ Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS) lungs
❑ Hektoen Enteric Agar (HEA) b.) Pericardial fluid - Found between pericardial sac
❑ Xylose Lysine Deoxycholate Agar (XLD) and heart
b) Enrichment c.) Peritoneal fluid - Found between peritoneal sac
❑ Enrichment and intestine
❑ Selenite F - Enrichment for Salmonella Shigella Agar
(SSA)
❑ Alkaline Peptone Water - Enrichment for Thiosulfate
Citrate Bile Salts Sucrose Agar (TCBS)
❑ Campylobacter Agar (CAMPY)

2. Microbiology Tests
a.) Microscopic
❑ Gram stain
❑ Acid Fast Bacilli stain
b.) Culture
❑ Bacterial culture
➢ Thioglycolate
➢ Chocolate Agar Plate (CAP)
❑ Mycobacterial culture - Lowenstein Jensen (LJ)
VI. GASTRIC SECRETION
medium
• Retrieved from the stomach
❑ Fungal culture - Sabouraud Dextrose Agar (SDA)
1. Possible Indications
❑ Viral culture - Cell / tissue culture
❑ Gastritis / Ulcer - Caused by Helicobacter pylori
❑ Pulmonary tuberculosis in children
VIII. SPECIMEN COLLECTED IN SWABS
2. Way of Collection
1. Specimens
❑ Intubation
❑ Rectal Swab
➢ Make use of hallow tube (orogastric tube,
➢ When stool is impossible to collect especially for
endotracheal tube)
pediatric patients
a.) Nasal - Nose – Larynx – Trachea – Stomach
➢ 3 – 4 cm onto the rectum and rotate
b.) Oral - Mouth – Larynx- Trachea – Stomach
➢ Culture Media
➢ Two Methods of Intubation / Aspiration
✓ Blood Agar Plate (BAP)
✓ Fractional - Aspirated every 15 mins up for 1
✓ Chocolate Agar Plate (CAP)
hr.
✓ MacConkey Agar (MAC)
✓ Complete - Aspirated continuous for 1 hr.
✓ Thioglycolate
3. Microbiology Tests
❑ Throat Swab
a.) Microscopic
➢ Where inflammation is visible
❑ Gram stain
➢ Placed in transgrow transport medium (amie’s,
❑ Acid Fast Bacilli stain
stuart’s, transgrow)
b.) Cultural
➢ Refrain from touching the tongue
❑ Bacterial culture - Thioglycolate - Chocolate
❑ Culture Media
Agar Plate (CAP)
✓ Blood Agar Plate (BAP)
❑ Mycobacterial culture - Lowenstein Jensen (LJ)
✓ Chocolate Agar Plate (CAP)
medium
✓ MacConkey Agar (MAC)
❑ Fungal culture - Sabouraud Dextrose Agar (SDA)
✓ Thioglycolate
❑ Viral culture - Cell / tissue culture
❑ Eye Swab
❑ Clinical Indications: Collected to diagnose
VII. ASPIRATED SAMPLE
ocular infections or inflammations, including
• Make use of syringe
conjunctivitis, corneal ulcers, or other eye
• Considered as sterile, both aerobic and anaerobic
infections.
can be processed
❑ Culture Media:

RDME | BSMT – 3C 8
 BACTERIOLOGY MLS – 110

LECTURE 5 - 6
✓ Blood Agar Plate (BAP) 2. Types of Swabs
❑ Rayon
✓ Chocolate Agar Plate (CAP) ❑ Dacron
✓ MacConkey Agar (MAC) ❑ Cotton
✓ Thioglycolate ❑ Calcium Alginate - Do not use for isolating virus for it
❑ Ear Swab may cause possible damage to cells
➢ Clinical Indications: Collected to ❑ Polyester
investigate ear infections, particularly otitis –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
externa, otitis media, or other ear-related PATHOGENIC ORGANISM
infections. 1. Rectal Swab:
➢ Culture Media ❑ Pathogenic Bacteria: Escherichia coli (including
✓ Blood Agar Plate (BAP) pathogenic strains like E. coli O157:H7), Shigella spp.,
✓ Chocolate Agar Plate (CAP) Salmonella spp., Campylobacter jejuni, Clostridium
✓ MacConkey Agar (MAC) difficile.
✓ Thioglycolate 2. Throat Swab:
❑ Nasal Swab ❑ Pathogenic Bacteria: Streptococcus pyogenes
➢ Clinical Indications: Used for detecting nasal (Group A Streptococcus), Corynebacterium
infections, such as rhinitis, sinusitis, or identifying diphtheriae (causing diphtheria), Neisseria
nasal colonization of pathogens like gonorrhoeae (in cases of pharyngeal gonorrhea),
Staphylococcus aureus (including MRSA) Haemophilus influenzae, Staphylococcus aureus.
➢ Culture Media 3. Eye Swab:
✓ Blood Agar Plate (BAP) ❑ Pathogenic Bacteria: Staphylococcus aureus,
✓ Chocolate Agar Plate (CAP) Streptococcus pneumoniae, Haemophilus
✓ MacConkey Agar (MAC) influenzae, Neisseria gonorrhoeae (in cases of
✓ Thioglycolate conjunctivitis in newborns), Pseudomonas
❑ Nasopharyngeal Swab aeruginosa.
➢ Culture Media 4. Ear Swab:
✓ Blood Agar Plate (BAP) ❑ Pathogenic Bacteria: Streptococcus pneumoniae,
✓ Chocolate Agar Plate (CAP) Haemophilus influenzae, Staphylococcus aureus
✓ MacConkey Agar (MAC) (including methicillin-resistant Staphylococcus
✓ Thioglycolate aureus - MRSA), Pseudomonas aeruginosa.
❑ Urethral Swab 5. Nasal Swab:
➢ For male, collected by the pathologist ❑ Pathogenic Bacteria: Staphylococcus aureus
➢ Placed in transgrow transport medium (including methicillin- resistant Staphylococcus
➢ Clinical Indications: Used to diagnose and aureus - MRSA), Streptococcus pneumoniae,
detect sexually transmitted infections (STIs) or Haemophilus influenzae, Corynebacterium species.
other urinary tract infections, particularly in cases 6. Nasopharyngeal Swab:
of suspected urethritis or STIs like chlamydia or ❑ Pathogenic Bacteria: Streptococcus pneumoniae,
gonorrhea. Haemophilus influenzae, Neisseria meningitidis,
➢ Culture Media Bordetella pertussis (causing whooping cough),
✓ Blood Agar Plate (BAP) Staphylococcus aureus.
✓ Chocolate Agar Plate (CAP) 7. Urethral Swab:
✓ MacConkey Agar (MAC) ❑ Pathogenic Bacteria: Neisseria gonorrhoeae,
✓ Thayer Martin Agar (THA) Chlamydia trachomatis, Mycoplasma genitalium,
✓ Modified Thayer Martin Agar Trichomonas vaginalis, Ureaplasma urealyticum.
❑ Vaginal Swab 8. Vaginal Swab:
➢ For female, collected by the OB/GYN ❑ Pathogenic Bacteria: Gardnerella vaginalis
➢ Placed in transgrow transport medium (associated with bacterial vaginosis), Candida
➢ Clinical Indications: Collected to diagnose species (e.g., Candida albicans causing yeast
vaginal infections, bacterial vaginosis, yeast infections), Neisseria gonorrhoeae, Chlamydia
infections (e.g., candidiasis), or sexually trachomatis, Trichomonas vaginalis.
transmitted infections (STIs) affecting the vaginal –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
area.
➢ Culture Media
✓ Blood Agar Plate (BAP)
✓ Chocolate Agar Plate (CAP)
✓ MacConkey Agar (MAC)
✓ Thayer Martin Agar (THA)
✓ Modified Thayer Martin Agar

RDME | BSMT – 3C 9