CLINICAL BACTERIOLOGY - LABORATORY

PRELIMS TOPIC 1. BACTERIAL MORPHOLOGY AND GRAM STAINING

Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT
August 11, 2023

BACTERIAL MORPHOLOGY
Staphylococci
● Refers to the form or shape of bacteria / – bunch of
microorganisms grapes, or like
● Germs and bacteria are the same. clusters/ in
○ Germ is the jargon word for bacteria bundle
● Are microorganisms bacteria?
○ Not all.
Staphylococcus spp.
THREE BASIC SHAPES OF BACTERIA (Staphylococcus
aureus)
1. Cocci
○ spherical in nature Sarcina
○ They can be oblong in shape, elongated,
or flattened on one end
○ Has a PLANE OF DIVISION
■ This is the way how they divide
themselves/ how they multiply or
replicate themselves
○ From the Greek word, cocci means berry.
cuboidal in shape or
like packets of 8
Coccus

2. Bacilli

● The plane of division is LIMITED

Diplococci ● “Little sticks”

Bacillus

Two

Tetrad
Diplobacilli

Plane of division
should be horizontal.

Neisseria spp.

Streptococci Streptobacilli

Bacilli in chains.

Palisades

Streptococcus spp.

Made by Miguel Astronomo and Daniel Budo 1

 ○ For Gram-positive microorganisms /
Coccobacilli
bacteria, they have a THICK
peptidoglycan layer.
■ Higher peptidoglycan content
■ Absorbs crystal violet
■ Gram’s Iodine helps form a
complex between it and Crystal
violet
● This will NOT be washed
Curve-Shaped off by the Acetone
Bacilli
alcohol due to its thick
peptidoglycan layer.
■ COLOR: VIOLET / PURPLE /
BLUE

○ For Gram-negative microorganisms /

Vibrio species
bacteria, they have a THIN peptidoglycan
layer.
3. Spiral ■ They also have very high lipid
● Helical / DNA-like content.
Divided into two ■ In the application of crystal violet,
● Spirilla it was absorbed.
○ “Corkscrew” ■ For Gram’s iodine, there was a
● Spirochete bridge.
○ Longer compared to spirilla ■ In Acetone alcohol decolorizer, it
was washed off because of its
Spirochete thin peptidoglycan layer.
■ Safranin came inside
■ COLOR: RED / PINK
● The basic principle of gram staining involves the
ability of the bacterial cell wall to retain the
crystal violet dye during solvent treatment
Treponema pallidum REAGENT OF GRAM STAINING
(agent of Syphilis) V Crystal Violet → Primary stain
Spirilla
I Gram’s Iodine → Mordant (bridge)
A Acetone Alcohol → Decolorizer
S Safranin → Counterstain (Secondary stain)

● Impart color to the structure

Spirillum minor
PURPOSE OF GRAM STAINING
(causative agent of
Rat bite fever) ● To differentiate gram-positive from gram-negative
organisms
● Since bacteria are complex and diverse, therefore
GRAM STAINING for us not be confused, Scientists divided them into
● Discovered in 1884 by Sir Hans Christian Gram two
● Differential staining
○ Gives contrast to differentiate varios
microorganisms
CONCEPT
● If the dye is basic, it will attach to the acidic
component of the cell
● If the dye is acidic, it will attach to the basic
component of the cell

● Opposite charges attract

● if both charges are the same, it will repel.

PRINCIPLE OF GRAM STAINING

● The reason behind the result of the test
● Based on the cell wall of bacteria
○ We have 2 cell walls present in the
bacteria

Made by Miguel Astronomo and Daniel Budo 2

 CLASSIFICATION OF BACTERIA IN STAINING
● For spiral, it needs special staining - fluorescent
stains
○ Cannot be stained using gram staining
1. GRAM-POSITIVE
● Almost all cocci
EXEMPTION
ALL COCCI ARE GRAM-POSITIVE, EXCEPT
(NeVerMind, Baby)
Neisseria
Veilonella
Moraxella
Branhamella
2. GRAM-NEGATIVE
● Almost all bacilli

ALL BACILLI ARE GRAM-NEGATIVE, EXCEPT

Mycobacterium
Corynebacterium
Clostridium
Bacillus
Erisypelothrix
Listera
Lactobacillus
Actinomyces
Nocardia
Gardnerella
Arcanobacterium

BACTERIAL SMEAR/ GRAM STAINING PROCEDURE

1. You can swab in different dirty compartments
2. After, you place the swab in the test tube, but make
sure the test tube has NSS inside.
3. Using the wire loop, heat it with the alcohol lamp for
sterilization.
4. Let it cool down.
5. Place the wire loop in the test tube
6. Spread the wire loop to the slide.
7. After spreading, get the slide (with the aid of
forceps) and let it pass through 3 times with the
alcohol lamp
8. Let it air dry, and apply the reagents
9. Crystal violet should be flooded for 1 minute
10. Gram’s Iodine for 1 minute
11. Acetone Alcohol for 10-15 seconds
12. Safranin for 30 seconds-1 minute
13. Every in between wash it off with running
water/distilled water, but be careful not to wash off
the smear. Run off the water on the topmost portion
of the slide.
14. Label the slide

Made by Miguel Astronomo and Daniel Budo 3

 CLINICAL BACTERIOLOGY – LABORATORY

PRELIMS TOPIC 2. ACID-FAST BACILLI STAINING

Lecturer/s / Source/s: Mr. Jason “Ab” Chua, RMT, MSMT, Dr. Aileen Grace Ang, RMT, MSBio, MSMT, Ms.
Joanne Krystine G. Tago, RMT, MAST-Bio, Ms. Doren Venus Otod, RMT
FULL TRANSES MASTERLIST: https://bit.ly/masterli_st

ACID-FAST BACILLI ○ Fluorescent microscope is needed

● Bacteria that “fasted” acid in which the bacilli, because fluorochrome is needed as the
specifically Mycobacteria reagent in order for us to perform this
○ Their cell wall have increased lipid method.
content which is Mycolic acid ○ FLUOROCHROME → impart light “fluoro,”
○ This is the reason why Mycobacteria and color “chrome”
resists acid.
It was only FRANZ ZIEHL who initially modified the stain
● This discovery was from Sir Robert Koch who
ACID-FAST STAINING discovered tubercle bacilli (Mycobacteria spp ;
● It is used to stain bacteria that have HIGH LIPID Mycobacterium tuberculosis)
contents in their cell walls. ● Tuberculosis was the leading cause of death
● It utilizes CARBOLFUCHSIN as the primary stain worldwide.
and METHYLENE BLUE or MALACHITE GREEN ● According to WHO in the year 2020, 9.9 million
as the secondary stain. have died due to tuberculosis–before that the
number was very high, not counting the
● In this procedure the cell wall of acid-fast bacteria
unreported cases.
resists the acid-alcohol (hydrochloric acid - ethanol ● Ziehl was curious about Sir Robert Koch’s
mixture) decolorization step discovery. He used Carbolfuchsin and heat –
● HEAT is applied as a mordant in the Ziehl Neelsen however this was not enough
Method while TERGITOL is used in the Kinyoun ○ This was then modified by Friedrich
Method. Neelsen who used Acid Alcohol
○ They figured out Mycobacteria have
increased lipid content
PRINCIPLE ○ This was then renamed to
● The primary stains binds to mycolic acid in the cell Ziehl-Neelsen method.
walls of acid fast bacteria and is retained after
decolorizing with acid alcohol
REAGENTS
Acid Fast Bacilli (AFB) RETAIN primary stain and deep-pink ZIEHL-NEELSEN METHOD
or red colored while non-AFB either blue or green colored. 1. Carbolfuchsin
● Primary stain
2. Heat
● Mordant
● After steam has come out of the slide,
leave it for 10 minutes.
3. Acid Alcohol
● Decolorizer
● 25-30 seconds
○ Mycobacteria will resist the
decolorization process of the Acid
Alcohol because of the increased
lipid content due to the Mycolic
acid present in the cell wall
○ Hence, the name “acid-fast”
TWO COMMON METHODS FOR AFB STAINING 4. Methylene Blue
● Ziehl-Neelsen Method ● 10 seconds
○ “Hot” Method ● Counterstain/Secondary Stain
○ Due to the physical mordant→ heat
● Kinyoun Method RESULTS
○ “Cold” Method
○ They replaced heat with AFB-positive RED/PINK
TERTIGOL/BLEACH as the physical (+) ● stay its primary stain
mordant. because they resisted Acid
○ THEY ONLY DIFFER IN MORDANTS VS. Alcohol (decolorizer)
ZIEHL-NEELSEN
● Auramine-Rhodamine Method AFB-negative BLUE/PURPLE/GREEN
● Lacks mycolic acid and allows
○ Not routinely used

Made by Miguel Astronomo and Daniel Budo 1

 8. Wash the slide with a gentle stream of running
the acid alcohol to wash out
carbolfuchsin, so the water.
counterstain comes in and 9. Tilt the slide to drain off excess rinsing water.
makes the microorganisms 10. Pour methylene blue to cover the whole surface of
blue/purple the slide and leave for 10 seconds.
SOURCE 11. Tilt the slide to drain off excess stain.
12. Wash the staining solution with a gentle stream of
PROCEDURE running water.
13. Tilt and place the slide on the slide rack to dry in the
Size of Template: 2 x 3 cm air. Don't place it under the sun to dry.
14. When the slide is dry, place a drop of immersion oil
on the stained smear. Do not touch the smear with
SPUTUM SMEAR PREPARATION
the dropper/stick to avoid transfer of from one
1. Prepare a clean glass slide. Label the specimen smear to another.
number at the frosted end of the slide. 15. Examine the smear under 100x objective with 10x
2. Fish out one loopful of yellowish particles of eyepiece. Acid-fast bacilli appear red or pink in
sputum. color, and the background is stained in blue color.
3. Spread one loopful of the sputum evenly on a clean
labeled glass slide, approximately 2 cm x 3 cm in
EVALUATION
size.
1. Read the slide systematically.
2. Do not read the smear in a zigzag manner because
this will increase the straining of the eyes. Proceed
from left or right end to the other end. Come down a
little and then proceed either from left to right (or
three vertical lines of the smear).
3. One horizontal line of a 3 cm width of the smear
corresponds to approximately 150 visual fields (VF)
of magnification x 1000 (100 x objective and 10 x
eyepiece).

NOTE: One may read along one imaginary horizontal line

from one end to the other of the smear if the reading is
NOTE: The rough end of the applicator stick can be used
positive (+n, 1+, 2+, 3+).
instead of a wire loop to take sputum to smear and the
pointed end may be used to make the smear. The used
4. Read two horizontal lines that correspond to 300
sticks are burnt after day's work.
visual fields to report the slide as negative.
5. Read at least one horizontal line. When AFB is
4. Allow the smear to dry completely at room
seen, count the number of bacilli present. But stop
temperature. Do not placed it under the heat of the
reading the smear when the average number of
sun orover a flame.
AFB in each field is 1 or more in less than one
5. After drying completely, fix it by passing through the
horizontal line and the smear is reported as (+++).
flame 2 to 3 times about 2-3 seconds each.
When AFB is not observed on one horizontal line,
Flame at the back of smeared surface of the slide.
continue to read second horizontal line.
Never scorch the smear. Blot dry with a paper towel
6. Report as Acid-Fast Bacilli and not such a comment
or issue.
as M. tuberculosis because the identification of
Mycobacterial species is not possible by
STAINING microscopic examination, but it is only possible after
1. Place the slide on a staining bridge/rack. cultivation of the bacilli.
Remember to leave enough space between slides
to prevent the transfer of material from one smear PROPER SCANNING
to another and the solution running off from the
slides.
2. Pour the Carbolfuchsin to cover the whole surface
of the slide
3. Heat the slide till steam comes off from the stain.
Do not boil and do not allow the slides to dry. Leave
it for 10 minutes.
4. Tilt the slide to drain off excess stain.
5. Wash the staining solution with a gentle stream of
running water.
6. Tilt the slide to drain off excess rinsing water. via Systematic Manner
7. Cover the whole slide with a decolorizer (acid From left to right (top) – 150 fields
alcohol) until the color no longer runs off the slide. From right to left (bottom) – 150 fields
This should take from 25 to 30 seconds ● Indicated with arrows

Made by Miguel Astronomo and Daniel Budo 2

 ● Horizontal Scanning MANTOUX TUBERCULIN TEST
● PPD → Purified Protein Derivative
- You can perform vertical scanning so as long as ○ Reagent
you reach 300 visual fields ● Skin test, injected in the arms
● Allergic Reaction
REPORTING FOR AFB-POSITIVE ○ (+) → TB
○ (-) → Negative for TB
NATIONAL STANDARD REPORTING SCALE FOR
ACID-FAST BASED ON THE DEPARTMENT OF
HEALTH’S MANUAL/GUIDELINE BAUMGARTEN’S STAIN
● Stain used for Colorblind individuals
0 No AFB seen in 300 visual fields ● Differentiates Mycobacterium tuberculosis from
Mycobacterium leprae
+n 1-9 AFB / 100 visual fields ○ M. leprae is the causative agent of leprosy

1+ 10-99 AFB / 100 visual fields DIGESTANT AND DECONTAMINANT

● Digestant
2+ 1-10 AFB / OIO in at least 50 visual fields
○ 2-4% NaOH
3+ > 10 AFB / OIO in at least 20 visual fields ■ Sputolysin
● Decontaminant
○ 2-4% NaOH
SPECIMEN REJECTION ■ Zephiran (Benzalkonium chloride)
BARTLETT’S CRITERIA
● If your sample is sputum or saliva COLLECTION
● <10 epithelial cells, >25 polymorphonuclears (PMN) ● First morning specimen
it is considered a SPUTUM ● 2-3 specimens needed
○ >10 epithelial cells, <25
polymorphonuclears → REJECT SAMPLE
because this is NOT SPUTUM (can be
saliva)
○ White Blood Cells should be numerous in
count due to infection - if the other way
around this would be contamination due to
abundance of epithelial cells

PCR GeneXpert
● Classifies TB as MDR-TB or XDR-TB

MDR-TB XDR-TB

→ Multi drug-resistant → Extremely drug-resistant

Tuberculosis Tuberculosis

Resistant to 2 drugs

FIRST GENERATION DRUGS (RIPES)

Rifampicin
Isoniazid
Pyrazinamide
Ethambutol
Streptomycin
SECOND GENERATION DRUGS (CCOKE)
Capriomycin
Ceprofloxacin
Ofloxacin
Kanamycin
Ethionamide

● These are drugs given to patients that have

tuberculosis.

Made by Miguel Astronomo and Daniel Budo 3

 CLINICAL BACTERIOLOGY - LABORATORY

PRELIMS TOPIC 4. BACTERIAL CULTIVATION / CULTURE MEDIA

Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT
FULL TRANSES MASTERLIST: https://bit.ly/masterli_st

CULTURE MEDIA / AGAR ○ Shiny, glistening surface

○ Staphylococcus
● Rough Colony
○ Dull, dry
○ Bacillus anthracis → medusa head
appearance

● Agar → also known as Agar-agar (Mucoid, smooth, rough)

○ We need to cultivate them “In vitro” - ● Condom-shaped colonies
outside of the body ○ Mycobacterium
○ We need to mimic the environment in CLASSIFICATION OF CULTURE MEDIA
order for them to grow, via “Cultivation” ACCORDING TO PHYSICAL STATE/ CONSISTENCY
○ Comes from Red algae
○ gelatin-like consistency
● Cultivation → cultivating microorganisms in vitro
● provides the appropriate biochemical and
biophysical environment for microbial growth
● Inoculum → material/substance to be inoculated in
the agar (e.g. Isolated colonies)
● Culture
○ Types: pure (virign/single species of 1. LIQUID – without any trace of agar or solidifying
organisms), substance (e.g., Nutrient broth) – placed in tube
○ mixed (more than 2 organisms), 2. SEMISOLID – contains 0.5-1.5% of agar (e.g.,
○ stock (for storage/maintained in order to Sulfide) → (SIM)
keep microorganisms viable/alive) 3. SOLID – contains 2-3% of agar (e.g., Blood Agar
Plate, Triple Sugar Iron)

Test Tube Brush appearance of Erysipelothrix rhusiopathiae

● any material containing the necessary nutritional on SIM (Sulfide indole motility). (Manner of inoculation →
and environmental requirements for bacterial Stab) (Wiere loop appearance)
growth
● CLASSIFICATION according to: PHYSICAL
STATE/ CONSISTENCY, COMPOSITION, HOW
THE MEDIUM IS DISPENSED AND USE
(function/purpose)
COLONY AND ITS TYPES
● Mucoid colony → sticky/wet
○ These organisms are being encapsulated
/presence of capsule
○ Klebsiella, Enterobacter
● Smooth Colony

Made by Miguel Astronomo, Daniel Budo 1

Inverted Umbrella/Christmas Tree appearance of Listeria
monocytogenes on Sulfide indole motility
ACCORDING TO COMPOSITION
● SYNTHETIC or DEFINED MEDIUM
○ Medium in which all components are
known (commercially prepared culture
media)
○ Preferred for the isolation of cyanobacteria
and chemoorganotrophs.
○ Example: BG 11 MEDIUM
● NON-SYNTHETIC OR COMPLEX MEDIUM 2. LYSINE IRON AGAR
○ Medium which some of the substances are ○ Stab-Streak-Stab
unknown (peptones, meat and yeast ■ Special form of Slant and butt
extracts) ○ Determines the ability of an organism to
○ Isolation of MEDICALLY IMPORTANT decarboxylate or deaminate in Lysine
BACTERIA → can cause diseases ○ Exception → 2 times stabbed (
○ Example: TSB, MAC
● TISSUE CULTURE MEDIUM
○ Obligate intracellular bacteria (Rickettsia
and Chlamydia)
○ Example: W138 CELLS, HeLa 229
(Henrietta Lacks) CELLS, MCCOY CELLS
ACCORDING TO DISPENSING OR DISTRIBUTION
● PLATED
○ Distributed in a sterile petri dish or plates
● TUBED
○ Tube media are prepared as either
LIQUID, SLANT, BUTT AND SLANT, or
BUTT 3. SIMMON’S CITRATE
○ Example: TRIPLE SUGAR IRON, LYSINE ○ Slant
IRON AGAR, SIMMONS' CITRATE, ○ Tests ability of bacteria to utilize citrate as
CHRISTENSEN UREA, SULFIDE sole source of carbon
INDOLE MOTILITY

MANNER OF INOCULATION

● Slant → zigzag
● Slant/ deep or Slant/ butt → zigzag(streak) and stab
● Butt/deep → stab
● Broth → stab or zigzag
● Broth + Durham tube → special technique;
water/food bacteriology
● Plate
ACCORDING TO DISPENSING OR DISTRIBUTION 4. CHRISTENSEN’S UREA AGAR
1. TRIPLE SUGAR IRON ○ Slant
○ Slant and butt ○ Pink - urease producers (positive)
○ Biochemical tests for Enteric bacteria or ○ Determines if bacteria has enzyme urease
Enterobacteriaceae that will react for Urea Agar
○ 3 sugars present → lactose, sucrose (both
1%) and glucose (0.1%)
○ Lactose and sucrose ➔ slant; Glucose ➔
butt

Made by Miguel Astronomo, Daniel Budo 2

 5. SULFIDE INDOLE MOTILITY ENRICHED
○ Deep Semi-solid
○ Stab
○ 3 tests for determination
■ Production of Sulfide (If tube has
black color, it is positive for
sulfide)
■ Indole (red rings in top →
positive)
■ Motility (turbid agar/ growth in
● Addition: blood, serum, egg, yolk, vitamins, AA
stab line → positive)
○ 5% sheep’s blood
○ Type O cells → if human blood is used so
there would be no antigens that would
cause lysis
○ Chocolate agar plates → blood in heat
causes lysis making its color brown
● Supports growth of fastidious organisms
● Solid culture bacteria
Examples: Blood agar plates, Chocolate agar plates,
Thayer-Martin

ENRICHMENT
FUNCTION/PURPOSE EXAMPLE

General purpose Nutrient agar

Enriched BAP (Blood agar plates), CAP

(Chocolate agar plates)

Enrichment TSB (Trypticase soy

broth/Tryptone soya broth),
Selenite

Selective EMB (Eosin-Methylene Blue

Agar), MacConkey
● Promotes growth of certain organisms
Differential EMB (Eosin-Methylene Blue ● Liquid culture bacteria
Agar), MacConkey, BAP Examples: Selenite F, BHI (Brain Heart Infusion),
(Blood Agar Plate) Thioglycollate
SELECTIVE
Anaerobic growth Thayer-Martin

Specimen transport Stuart

Assay MHA (Mueller Hinton Agar)

Enumeration PDA (Potato Dextrose Agar)

GENERAL PURPOSE MEDIA

● contains inhibitory agents that prevents / suppress

growth of unwanted microbes
● permits preliminary identification of genus or spp.
Examples: Mannitol Salt Agar, Hektoen Enteric Agar
Thayer-Martin → has Vancomycin to inhibit gram-positive
bacteria; Histatin to inhibit fungi, etc.
MEDIUM SELECTIVE USES
AGENT

BASAL MEDIA Mueller Tellurite Potassium Isolation of

● Supports most non-fastidious bacteria tellurite Corynebacterium
● for primary isolation of microorganism diphtheria
● Designed to grow a broad spectrum of microbes
Examples: nutrient agar, nutrient broth Enterococcus Na azide Isolation of fecal
faecalis broth Tetrazolium Entercocci

Made by Miguel Astronomo, Daniel Budo 3

 Phenylethanol Phenylethanol Isolation of
agar chloride Staphylococcus
spp. and
Streptococcus
spp.

Tomato juice Tomato juice, Isolation of

agar acid Lactobacilli

MacConkey Bile, crystal violet Isolation of Gram

(-) enteric THREE HEMOLYTIC PATTERNS
1. beta hemolysis → complete hemolysis (yellow)
SSA (Samonella Bile, citrate, Isolation of 2. alpha hemolysis → incomplete/partial hemolysis
Shigella Agar) brilliant green Salmonella & 3. gamma hemolysis → no hemolytic pattern
dye Shigella
MEDIUM Substance for Differentiates
Lowenstein-Jens Malachite green Isolation & differentiation between
en agar dye maintenance of
Mycobacteria BAP (Blood Intact RBCs Types of
Agar Plate) hemolysis
Sabouraud’s pH 5.6 Isolation of fungi
agar MSA (Mannitol Mannitol, phenol Species of
salt agar) red, 7.5% NaCl Staphylococcus

DIFFERENTIAL HEA (Hektoen Bromthymol blue, Salmonella,

enteric agar) salicin, lactose, Shigella, other
sucrose, sodium LFs and NLFs
thiosulfate, ferric
ammonium
citrate, bile

SBA (Sheep Spirit blue dye, Fat-utlizing

Blood Agar) oil bacteria from
those that do not

Urea broth Urea, phenol red Urea-hydrolyzing

● displays visible differences among microbes bacteria
● Indicators
Examples: SIM (Sulfide Thiosulfate, Fe H2S production
● MacConkey agar indole motility) (Iron)
○ Neutral red = indicates lactose
fermentation (yellow) – negative for lactose TSI (Triple Triple sugars, Fe Sugar
fermentation (pink) Sugar Iron) (Iron), phenol red fermentation &
dye H2S production

XLD agar Xylose, lysine, Allows

(Xylose Lysine lactose, sucrose, differentiation of
Deoxycholate phenol red, xylose
Agar) sodium fermentation,
deoxycholate lysine
decarboxylation
& H2S
production

SPECIMEN TRANSPORT

CARY-BLAIR
● Spirit blue agar
○ Spirit blue dye = indicates fat hydrolysis
AMIES
○ bacteria that has lipase enzymes TRANSGROWTH
DIFFERENTIAL MEDIA
STUART
● Used for specimens that are sensitive for drying
● Make it easy to distinguish colonies of different
ASSAY
microbes
● Mueller-Hinton agar (MHA) → used for AST
[Antimicrobial Susceptibility Testing] to determine if
bacteria is drug-resistant or drug-susceptible
ENUMERATION
● Potato Dextrose Agar→ used for yeasts and
molds for counting

Made by Miguel Astronomo, Daniel Budo 4

 CLINICAL BACTERIOLOGY - LABORATORY

PRELIMS TOPIC 4. BACTERIAL CULTIVATION / CULTURE MEDIA

Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT
FULL TRANSES MASTERLIST: https://bit.ly/masterli_st

CULTURE MEDIA / AGAR ○ Shiny, glistening surface

○ Staphylococcus
● Rough Colony
○ Dull, dry
○ Bacillus anthracis → medusa head
appearance

● Agar → also known as Agar-agar (Mucoid, smooth, rough)

○ We need to cultivate them “In vitro” - ● Condom-shaped colonies
outside of the body ○ Mycobacterium
○ We need to mimic the environment in CLASSIFICATION OF CULTURE MEDIA
order for them to grow, via “Cultivation” ACCORDING TO PHYSICAL STATE/ CONSISTENCY
○ Comes from Red algae
○ gelatin-like consistency
● Cultivation → cultivating microorganisms in vitro
● provides the appropriate biochemical and
biophysical environment for microbial growth
● Inoculum → material/substance to be inoculated in
the agar (e.g. Isolated colonies)
● Culture
○ Types: pure (virign/single species of 1. LIQUID – without any trace of agar or solidifying
organisms), substance (e.g., Nutrient broth) – placed in tube
○ mixed (more than 2 organisms), 2. SEMISOLID – contains 0.5-1.5% of agar (e.g.,
○ stock (for storage/maintained in order to Sulfide) → (SIM)
keep microorganisms viable/alive) 3. SOLID – contains 2-3% of agar (e.g., Blood Agar
Plate, Triple Sugar Iron)

Test Tube Brush appearance of Erysipelothrix rhusiopathiae

● any material containing the necessary nutritional on SIM (Sulfide indole motility). (Manner of inoculation →
and environmental requirements for bacterial Stab) (Wiere loop appearance)
growth
● CLASSIFICATION according to: PHYSICAL
STATE/ CONSISTENCY, COMPOSITION, HOW
THE MEDIUM IS DISPENSED AND USE
(function/purpose)
COLONY AND ITS TYPES
● Mucoid colony → sticky/wet
○ These organisms are being encapsulated
/presence of capsule
○ Klebsiella, Enterobacter
● Smooth Colony

Made by Miguel Astronomo, Daniel Budo 1

Inverted Umbrella/Christmas Tree appearance of Listeria
monocytogenes on Sulfide indole motility
ACCORDING TO COMPOSITION
● SYNTHETIC or DEFINED MEDIUM
○ Medium in which all components are
known (commercially prepared culture
media)
○ Preferred for the isolation of cyanobacteria
and chemoorganotrophs.
○ Example: BG 11 MEDIUM
● NON-SYNTHETIC OR COMPLEX MEDIUM 2. LYSINE IRON AGAR
○ Medium which some of the substances are ○ Stab-Streak-Stab
unknown (peptones, meat and yeast ■ Special form of Slant and butt
extracts) ○ Determines the ability of an organism to
○ Isolation of MEDICALLY IMPORTANT decarboxylate or deaminate in Lysine
BACTERIA → can cause diseases ○ Exception → 2 times stabbed (
○ Example: TSB, MAC
● TISSUE CULTURE MEDIUM
○ Obligate intracellular bacteria (Rickettsia
and Chlamydia)
○ Example: W138 CELLS, HeLa 229
(Henrietta Lacks) CELLS, MCCOY CELLS
ACCORDING TO DISPENSING OR DISTRIBUTION
● PLATED
○ Distributed in a sterile petri dish or plates
● TUBED
○ Tube media are prepared as either
LIQUID, SLANT, BUTT AND SLANT, or
BUTT 3. SIMMON’S CITRATE
○ Example: TRIPLE SUGAR IRON, LYSINE ○ Slant
IRON AGAR, SIMMONS' CITRATE, ○ Tests ability of bacteria to utilize citrate as
CHRISTENSEN UREA, SULFIDE sole source of carbon
INDOLE MOTILITY

MANNER OF INOCULATION

● Slant → zigzag
● Slant/ deep or Slant/ butt → zigzag(streak) and stab
● Butt/deep → stab
● Broth → stab or zigzag
● Broth + Durham tube → special technique;
water/food bacteriology
● Plate
ACCORDING TO DISPENSING OR DISTRIBUTION 4. CHRISTENSEN’S UREA AGAR
1. TRIPLE SUGAR IRON ○ Slant
○ Slant and butt ○ Pink - urease producers (positive)
○ Biochemical tests for Enteric bacteria or ○ Determines if bacteria has enzyme urease
Enterobacteriaceae that will react for Urea Agar
○ 3 sugars present → lactose, sucrose (both
1%) and glucose (0.1%)
○ Lactose and sucrose ➔ slant; Glucose ➔
butt

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 5. SULFIDE INDOLE MOTILITY ENRICHED
○ Deep Semi-solid
○ Stab
○ 3 tests for determination
■ Production of Sulfide (If tube has
black color, it is positive for
sulfide)
■ Indole (red rings in top →
positive)
■ Motility (turbid agar/ growth in
● Addition: blood, serum, egg, yolk, vitamins, AA
stab line → positive)
○ 5% sheep’s blood
○ Type O cells → if human blood is used so
there would be no antigens that would
cause lysis
○ Chocolate agar plates → blood in heat
causes lysis making its color brown
● Supports growth of fastidious organisms
● Solid culture bacteria
Examples: Blood agar plates, Chocolate agar plates,
Thayer-Martin

ENRICHMENT
FUNCTION/PURPOSE EXAMPLE

General purpose Nutrient agar

Enriched BAP (Blood agar plates), CAP

(Chocolate agar plates)

Enrichment TSB (Trypticase soy

broth/Tryptone soya broth),
Selenite

Selective EMB (Eosin-Methylene Blue

Agar), MacConkey
● Promotes growth of certain organisms
Differential EMB (Eosin-Methylene Blue ● Liquid culture bacteria
Agar), MacConkey, BAP Examples: Selenite F, BHI (Brain Heart Infusion),
(Blood Agar Plate) Thioglycollate
SELECTIVE
Anaerobic growth Thayer-Martin

Specimen transport Stuart

Assay MHA (Mueller Hinton Agar)

Enumeration PDA (Potato Dextrose Agar)

GENERAL PURPOSE MEDIA

● contains inhibitory agents that prevents / suppress

growth of unwanted microbes
● permits preliminary identification of genus or spp.
Examples: Mannitol Salt Agar, Hektoen Enteric Agar
Thayer-Martin → has Vancomycin to inhibit gram-positive
bacteria; Histatin to inhibit fungi, etc.
MEDIUM SELECTIVE USES
AGENT

BASAL MEDIA Mueller Tellurite Potassium Isolation of

● Supports most non-fastidious bacteria tellurite Corynebacterium
● for primary isolation of microorganism diphtheria
● Designed to grow a broad spectrum of microbes
Examples: nutrient agar, nutrient broth Enterococcus Na azide Isolation of fecal
faecalis broth Tetrazolium Entercocci

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 Phenylethanol Phenylethanol Isolation of
agar chloride Staphylococcus
spp. and
Streptococcus
spp.

Tomato juice Tomato juice, Isolation of

agar acid Lactobacilli

MacConkey Bile, crystal violet Isolation of Gram

(-) enteric THREE HEMOLYTIC PATTERNS
1. beta hemolysis → complete hemolysis (yellow)
SSA (Samonella Bile, citrate, Isolation of 2. alpha hemolysis → incomplete/partial hemolysis
Shigella Agar) brilliant green Salmonella & 3. gamma hemolysis → no hemolytic pattern
dye Shigella
MEDIUM Substance for Differentiates
Lowenstein-Jens Malachite green Isolation & differentiation between
en agar dye maintenance of
Mycobacteria BAP (Blood Intact RBCs Types of
Agar Plate) hemolysis
Sabouraud’s pH 5.6 Isolation of fungi
agar MSA (Mannitol Mannitol, phenol Species of
salt agar) red, 7.5% NaCl Staphylococcus

DIFFERENTIAL HEA (Hektoen Bromthymol blue, Salmonella,

enteric agar) salicin, lactose, Shigella, other
sucrose, sodium LFs and NLFs
thiosulfate, ferric
ammonium
citrate, bile

SBA (Sheep Spirit blue dye, Fat-utlizing

Blood Agar) oil bacteria from
those that do not

Urea broth Urea, phenol red Urea-hydrolyzing

● displays visible differences among microbes bacteria
● Indicators
Examples: SIM (Sulfide Thiosulfate, Fe H2S production
● MacConkey agar indole motility) (Iron)
○ Neutral red = indicates lactose
fermentation (yellow) – negative for lactose TSI (Triple Triple sugars, Fe Sugar
fermentation (pink) Sugar Iron) (Iron), phenol red fermentation &
dye H2S production

XLD agar Xylose, lysine, Allows

(Xylose Lysine lactose, sucrose, differentiation of
Deoxycholate phenol red, xylose
Agar) sodium fermentation,
deoxycholate lysine
decarboxylation
& H2S
production

SPECIMEN TRANSPORT

CARY-BLAIR
● Spirit blue agar
○ Spirit blue dye = indicates fat hydrolysis
AMIES
○ bacteria that has lipase enzymes TRANSGROWTH
DIFFERENTIAL MEDIA
STUART
● Used for specimens that are sensitive for drying
● Make it easy to distinguish colonies of different
ASSAY
microbes
● Mueller-Hinton agar (MHA) → used for AST
[Antimicrobial Susceptibility Testing] to determine if
bacteria is drug-resistant or drug-susceptible
ENUMERATION
● Potato Dextrose Agar→ used for yeasts and
molds for counting

Made by Miguel Astronomo, Daniel Budo 4

 CLINICAL BACTERIOLOGY – LABORATORY

PRELIMS TOPIC 5. Use of Colony Morphology for Presumptive Identification of

Microorganisms
Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT
FULL TRANSES MASTERLIST: https://bit.ly/masterli_st

IMPORTANCE OF COLONIAL MORPHOLOGY GROSS COLONY CHARACTERISTICS

● Provide a presumptive identification to the physician 1. HEMOLYTIC PATTERN
● Enhances the quality of patient care through rapid
reporting of results and by increasing the On Sheep Blood Agar, hemolysis (Greek hemo: pertaining to
cost-effectiveness of laboratory testing. red blood cells [RBCs]; lysis: dissolution or break apart)
○ Serologic tests are extremely expensive observed in the medium immediately surrounding or
● Play a significant role in quality control, especially of underneath the colony is a reaction caused by enzymatic or
automated procedures and other commercially toxin activity of bacteria.
available identification procedures. ● Hemolysis (e.g., a, B, or no hemolysis with other
colony characteristics) on SBA is helpful in
presumptive identification, particularly of
streptococci and enterococci.
● It is important to determine whether true hemolysis
is present or whether discoloration of the medium is
the result of the growth of the organism on the
plate. Often the colony has to be removed with a
loop to visualize the hemolytic pattern.
● Proper technique requires the passing of bright light
through the bottom of the plate (transillumination) to
determine whether the organism is hemolytic (Fig.
8.5). Many organisms have no lytic effect on the
RBCs in Sheep Blood Agar and are referred to as
nonhemolytic.

A ➔ beta-hemolysis, B ➔ alpha hemolysis, C ➔ None

(gamma)

ALPHA HEMOLYSIS
● Incomplete hemolysis
● Characterized by GREENISH ZONES around the
colonies
● Example: Streptococcus pneumoniae

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➔ a-Hemolysis is partial lysing of RBCs in an SBA plate
around and under the colony that results in a green
discoloration of the medium. Examples of organisms that
produce a-hemolysis include Streptococcus pneumonia and
certain viridans streptococci.

BETA HEMOLYSIS
● Complete lysis
● Characterized by CLEAR ZONES around the
colonies
● Example: Streptococcus pyogenes

➔ B-Hemolysis is complete clearing of erythrocytes in SBA

around or under the colonies because of the complete lysis
of RBCs. Certain organisms, such as group A B-hemolytic
streptococci (Streptococcus pyogenes), produce a wide,
deep, clear zone of B-hemolysis,
● whereas others, such as group B B-hemolytic
streptococci (Streptococcus agalactiae) and Listeria
monocytogenes (a gram- positive rod) produce a
narrow, diffuse zone of B-hemolysis close to the
colony.
These features are helpful hints in the identification of certain
bacteria species

DOUBLE ZONE OF HEMOLYSIS

● Found in Clostridium perfringens
● Characterized by BETA HEMOLYTIC inner zone of
hemolysis due to production of theta toxin and in
ALPHA HEMOLYTIC outer zone of hemolysis due
to the presence of alpha toxin a.k.a lecithinase

2. FORM/SHAPE/MARGIN

● Description of the shape of colonies, as well as the

edges of the colonies
● Can be described as smooth, filamentous, rough
or rhizoid or irregular

DESCRIPTIONS
Bacillus anthracis MEDUSA HEAD COLONIES;
LION FACE

Proteus spp. SWARMING OF COLONIES

Proteus mirabilis – swarms on
nonselective agar, such as blood
or chocolate agar plate.

Diphtheroid ROUGH COLONIES

Yeast STARS OR COLONIES WITH

FEET OR PEDICLES

Pseudomonas SERRATED
aeruginosa

Actinomyces MOLAR TOOTH COLONIES

israelli

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 3. ELEVATION
● Determined by tilting the plate and looking at the 5. SIZE
sidd of the colony ● Colonies can be described as LARGE, MEDIUM,
● Described as flat/circular, raised, convex, SMALL
umbilicate crateriform, umbonate ● Streptococcus pyogenes have PINPOINT
● Streptococcus pneumonia are usually umbilicate COLONIES
● Viridans are umbonate ● Staphylococcus aureus have PINHEAD COLONIES
● Beta hemolytic Streptococci are generally flat

➔ Colonies are described as large, medium, small, or

pinpoint. However, a microbiologist rarely takes a ruler and
actually measures a colony. Size is generally a visual
comparison between genera or species. For example, gram-
positive bacteria generally produce smaller colonies than
gram-negative bacteria. Staphylococcus species are usually
larger than Streptococcus species.

6. CONSISTENCY/TEXTURE
● Determined by touching the colony with a sterile
loop
● Can be described as:
➔ The elevation should be determined by tilting the culture
○ Brittle, crumbly or wrinkled = Nocardia spp.
plate and looking at the side of the colony.
○ Creamy = Staphylococcus aureus
● Elevation may be raised, convex, flat, umbilicate
○ Sticky = some Neisseria, Klebsiella
(depressed center, concave), or umbonate (raised
○ Dry or Waxy = Diphtheroids
or bulging center, convex). S. pneumonia typically
produces umbilicate colonies, unless the colonies
➔ Consistency is determined by touching the colony with a
are mucoid because of the presence of a
sterile loop. Colony consistency may be brittle (splinters),
polysaccharide capsule. S. aureus typically
creamy (butyrous), dry, or waxy; occasionally, the entire
produces convex colonies. In comparison,
colony adheres (sticks) to the loop. S. aureus is creamy,
B-hemolytic streptococci generally produce flat
whereas certain Neisseria spp. are sticky. Nocardia spp.
colonies.
produce colonies that are brittle, crumbly, and wrinkled,
resembling bread crumbs on a plate. Diphtheroid colonies
are usually dry and waxy. Most -hemolytic streptococci are
4. DENSITY/OPTICAL PROPERTY
dry (except for mucoid types), and when pushed by a loop,
● Colonies are TRANSPARENT if they allow all the
the whole colony remains intact.
light to pass through
● Colonies are TRANSLUCENT if they allow some of
the light to pas through; Streptococcus agalctiae
7. COLOR
● Colonies are OPAQUE if they do not allow the light
● Colonies can be described as WHITE, GRAY,
to pass through; Staphylococcus aureus
YELLOW/BUFF
● Bordetella pertussis is described as a shiny, like a
● Cons are WHITE
half pear on blood containing media
● Enterococcus are usually gray
● Diphtheroids are buff colored

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➔ In contrast to pigmentation, color is a term used to
describe a particular genus in general. Colonies may be
white, gray, yellow, or buff. Coagulase-negative
staphylococci are white, whereas Enterococcus spp. may
appear gray. Certain Micrococcus spp. and Neisseria spp.
(nonpathogenic) are yellow or off- white. Diphtheroids are
buff. Most gram-negative rods are gray on SBA.

8. PIGMENT
● Inherent characteristic of a specific organism
confined generally to the colony
● Pseudomonas aeruginosa = blue green (pyocyanin
- pyoverdin)
● Serratia marcescens = red (prodigiosin)
● Kluyvera = blue
● Chromobacterium violaceum = violet (violacein)
● Provotella malaninogenica = brown - black

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