By: Fran Siska

Moderator: dr. Anik Widijanti, Sp. PK (K)

CULTIVATION
Cultivation :

The process of propagating organisms by taking

bacteria from the infection site by some means of
specimen collection and growing them in providing
the artificial / proper environmental conditions.

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Purpose:
 To demonstrate the presence of organisms which may be
causing disease.
 To determine which of the bacteria that grow are most
likely causing infection and which are likely contaminants or
colonizer.
 To obtain sufficient growth of clinically relevant bacteria to
allow identification, characterization, and susceptibility
testing.
 To test the susceptibility of pathogens to antimicrobial
agent
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Environmental factors affecting growth :
• Nutrients
• pH
• Temperature
• Aeration
• Salt concentration
• Ionic strength of the medium

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Nutrients
• Hydrogen donors and acceptors : ±2 g/L
• Carbon source : ±1 g/L
• Nitrogen source : ±1 g/L
• Minerals : sulfur and phosphorus, @±50 mg/L, and
trace elements, @0.1–1 mg/L
• Growth factors : amino acids, purines, pyrimidines,
@±50 mg/L, and vitamins, @0.1–1 mg/L
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pH
• The optimal pH must be empirically determined
for each species
• Most organisms (neutralophiles) grow best at
6.0–8.0
• Acidophiles have optimal as low as pH 3.0
• Alkaliphiles have optimal as high as pH 10.5

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Temperature
• Different microbial species vary widely in their
optimal temperature ranges for growth
• Psychrophilic forms grow best at 15–20 °C
• Mesophilic forms grow best at 30–37 °C
• Thermophilic forms grow best at 50–60 °C
• Some organisms are hyper thermophilic and can
grow at well above the temperature of boiling water
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Aeration
• Obligate aerobes : specifically requiring oxygen
as hydrogen acceptor
• Facultative : able to live aerobically or
anaerobically
• Obligate anaerobes : requiring a substance other
than oxygen as hydrogen
acceptor and being sensitive
to oxygen inhibition
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Ionic strength & Osmotic Pressure
• Halophilic: organisms requiring high salt
concentrations
• Osmophilic: organisms requiring high osmotic
pressures

Osmolality is regulated by the active transport of K+

ions into the cell

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Factors affecting the choice of culture media
• The major pathogens to be isolated  their growth
requirements and the recognizing features
• Source  steriles sites or from sites having a normal
flora
• Cost, availability, and stability of different media
• Training and experience of laboratory staff

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 The main type of culture media
Basic • simple media for microorganisms
without special nutritional
requirements
• e.g.: nutrient agar and nutrient broth

Nutrient Agar Nutrient Broth

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 The main type of culture media

Enriched • for microorganisms with exacting growth

requirements
• Enrichment media: fluid selective media
which contain substances that inhibit the
growth of unwanted organisms
• E.g.: Rappaport-Vassiliadis broth

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Rappaport-Vassiliadis broth

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 The main type of culture media

Selective • solid media which contain substances

which inhibit the growth of one
organism to allow the growth of another
to be more clearly demonstrated
• used when culturing a specimen from a
site having normal flora
• e.g.: TCBS agar

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16
 The main type of culture media
Indicator • a media to which dyes or other substances
are added to differentiate microorganisms
• e.g.: MacConkey agar

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 The main type of culture media
Transport • mostly semisolid media that contain
ingredients to prevent the overgrowth of
commensals and ensure the survival of
aerobic and anaerobic pathogens when
specimens cannot be cultured immediately
after collection
• e.g.: Cary-Blair medium and Amies transport
medium
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 The main type of culture media
Identification • include media to which substrates or
chemicals are added to help identify
bacteria isolated on primary cultures
• e.g.: peptone water sugars, urea broth,
and Kligler iron agar

Peptone water sugar Urea broth Kligler iron agar 19

 Culture media by consistency
Solid • Media are solidified by incorporating a gelling agent
such as agar or gelatin
• Used mainly in petri dishes as plate cultures. Also in
bottles or tubes as stab (deeps) or slope cultures
Semi-solid • prepared by adding a small amount of agar (0.4–
0.5% w/v) to a fluid medium
• used mainly as transport media, and for motility and
biochemical tests.
Fluid • most commonly used as enrichment where
organisms are likely to be few e.g. blood culture
• may also be used for biochemical testing e.g.
peptone water sugars or the use of media containing
tryptophan to detect indole production by some
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enterobacteria
• Liquid (broth) • Solid (agar)
 Strict anaerobes  bottom

 Aerobes  near the surface

 Microaerophilic  slightly below the surface

 Facultative anaerobes & aerotolerant 

throughout the medium

 Blood agar
 Brain heart infusion agar or broth agar
 Chocolate agar
 Columbia CNA with blood
 Gram-negative (GN) broth
 Hektoen Enteric (HE) agar
 MacConkey agar
 Phenylethyl Alcohol (PEA) agar
 Modified Thayer-Martin (MTM) agar
 Thioglycollate broth
 Xylose-Lysine-Desoxycholate (XLD) agar
1. Prepare media (dehydrated products) in damp-free
environment & wear a dust mask
2. Wash the hands immediately after preparing media
3. Do not delay making up the medium after weighing
4. Use completely clean equipment
5. Use distilled water or deionized water
6. Mixing the powder by stirring or rotating the
container (≠shake)

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7. Overheating a medium will alter its nutritional,
gelling properties & pH
8. Autoclave a medium only when the ingredients are
completely dissolved
9. Dispense medium in bottles or tubes in amounts
convenient for use
10. Know the length of time prepared-media can be
stored without deteriorating

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Aseptic techniques
• Flame sterilize wire loops, straight wires & metal
forceps before & after use
• Flame the necks of specimen bottles, culture bottles
& tubes after removing and before replacing caps,
bungs, plugs
• Avoid tops or caps of bottles & tubes touch an
unsterile surface

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Aseptic techniques

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Innoculation of media in petri dishease
• medium must be dried  30’-40’incubation at 35-37°C
• must provide single colonies for identification
• not necessary to use whole plates for every specimen

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Innoculation of slopes
• Slopes: Dorset egg medium or
Loeffler serum
• Use a sterile straight wire
• Streaking down at the centre &
then spread the inoculum in a
zig-zag pattern
• Kligler iron agar  inoculate a
slope and butt medium
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Innoculation of stab media
• Use a sterile straight wire to inoculate a stab
medium
• Stab through the centre of the medium

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Innoculation of fluid media
• using a sterile wire loop, straight wire, or Pasteur
pipette
wire loop : hold the bottle or tube at an angle and
rub the loop against the side of the container below
the level of the fluid

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• Incubated as soon as possible
- affect the viability of pathogens
- increase the risk of contamination (insects & dust)
• Incubation temperature (35-37°C), humidity & gaseous
atmosphere: suitable with their metabolism
• Length of time of incubation depends on how long to
develop the recognized cultural characteristics

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• Helps to differentiate pathogens
• Several techniques for obtaining anaerobic conditions:
- sachets containing oxygen-removing chemicals
- Copper coated steel wool to remove oxygen
- Reducing agents in culture media

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+2

+3–
4
 Size [mm or relative terms (pinpoint, small, medium,
large)]
 Pigmentation
 Shape (form, elevation, margin)
 Surface appearance (glistening, opaque, dull, dry,
tranparent)
 Change in agar media
 Odor

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STAINING
Staining
An auxiliary technique that used to better visualize cells and
cell components under a microscope
Purpose:
- to get a rapid presumptive diagnosis of an infection
- to help to identify a pathogen  To know the
morphology, structure, characteristic the pathogen

To provide reliable information, smears must be prepared,

labelled, and fixed correctly prior to being stained!! 51
 Smear preparation varies depending on the type of
specimen being processed and on the staining
methods to be used.
 General rule for smear preparation:
Sufficient material must be applied to the slide so
that chances for detecting and distinguishing
microorganisms are maximized
Avoid the application of excessive material
The staining method to be used is dictated by
which microorganisms are suspected in the
specimen.
 To provide reliable information  prepared,
labeled, & fixed correctly
 Labeling slides
 date, the patient’s name & number
 slides with a frosted end for labeling
 use lead pencil
 slides from (+) AFB smears  discarded and
never reused
 scratched, chipped, and discolored slides 
discarded
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 Smears should be spread evenly covering an
area (d:15–20 mm)
 Precautions in handling infectious material
 Different specimens  different techniques

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Purulent specimen
Sterile wire loop, make a thin preparation
Non-purulent fluid specimen
Centrifuge the fluid & make a smear from
a drop of the well-mixed sediment

Culture
Emulsify a colony in sterile distilled water,
make a thin preparation on a slide.
If a broth culture, transfer a loopfull to a
slide and make a thin preparation
Sputum
Use a piece of clean stick to transfer, spread purulent
and caseous material on a slide
Soak the stick in a phenol or hypochlorite disinfectant
before discarding it
Swabs
Roll the swab on a slide (avoids damaging the pus
cells)
Particularly important to search intracellular bacteria
such as N. gonorrhoeae (urethral, cervical, or eye
swab)
Feces
Use a piece of clean stick to transfer to a slide
Spread to make a thin preparation
Decontaminate the stick before discarding it
 Leave the slide in a safe place for the smear to air-dry 
protected from dust, flies, cockroaches, ants, and direct
sunlight
 If requires urgent staining, it can be dried quickly using
gentle heat
 Smears taken from in-patients and during out-patient
clinics must always be transported to the laboratory in a
covered container
 Fixation is to preserve microorganisms & prevent smears
being washed during staining
 Smears are fixed by heat, alcohol, or by other chemicals 57
Heat fixation
 Widely used, excessive heat  damage
organisms & alter their staining reactions
 Also damages leucocytes  unsuitable for
fixing smear with intracellular organisms (N.
gonorrhoeae & N. meningitidis)
 Recommended technique:
1.Allow the smear to air-dry completely
2.Rapidly pass the slide, smear uppermost, 3x
through the flame of a spirit lamp or pilot flame of a
Bunsen burner
3.Allow the smear to cool before staining it
Alcohol fixation
 Far less damaging to microorganisms
 Cells (esp. pus cells) are well preserved
 Recommended for fixing smears with gram negative
intracellular diplococci
 Alcohol fixation is more bactericidal than heat
 The technique:
1. Allow the smear to air-dry completely
2. For the detection of intracellular gram (-) diplococci  1-2 drops
of absolute methanol or ethanol. For other organisms including M.
tuberculosis, fix with 1-2 drops of 70% v/v methanol or ethanol
3. Leave the alcohol on the smear for a min 2’ or until the alcohol
evaporates
OTHER CHEMICALS
 Sometimes necessary to fix smears which
contain particularly dangerous organisms to
ensure all the organisms are killed
 Ex : 40 g/l potassium permanganate for
anthrax bacilli

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Precautions
• Use a staining rack
• Do not attempt to stain a smear that is too thick
• To dispense stains, alcoholic and acetone reagents,
use dropper bottles or other spouted containers that
can be closed between use
• Label clearly stains and reagents
• Follow exactly a staining technique, particularly
staining and decolorizing times
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Precautions
• When washing smears of specimens which can be
easily washed from a slide, direct the water from a
wash bottle on the back of the slide, not directly on
the smear
• After staining, place the slides at an angle in a
draining rack for the smears to air-dry
• To check staining results, use quality control smears
of organisms, particularly when a new batch of stain
is used 62
Staining techniques

• Gram • Albert staining of volutin

• Ziehl-Neelsen (ZN)
granules
• Giemsa
• Auramine-phenol
• Acridine orange
• Methylene blue
fluorochrome
• Wayson’s bipolar

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  Purpose : To help identify pathogens in specimens &
cultures by their Gram reaction (+/-) & morphology
 Gram reaction due to permeability of the cell wall
during the staining process
Reagent
Crystal violet stain primary dye
(gentian or methyl)
Gram’s Iodine, a stabilizes the Crystal violet into the
mordant peptidoglycan layer
Acetone–alcohol decolorization, dissolves lipids (gram
decolorizer negative)
Safranin, neutral red counterstain
1 min 1 min 5-15 sec 1 min
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Procedure:
1. Fix smear by heat or using methanol
2. Cover with crystal violet
3. Wash with water. Do not blot
4. Cover with Gram’s iodine
5. Wash with water. Do not blot
6. Decolorize for 10-30 seconds with gentle
agitation in acetone (30 ml) and alcohol (70 ml)
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Procedure:
7. Wash with water. Do not blot
8. Cover for 10-30 seconds with safranin (2,5%
solution in 95% alcohol)
9. Wash with water and let dry.

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REPORTING GRAM SMEARS:
• Numbers of bacteria present (many, moderate, few or
scanty)
• Gram reaction : Gram (+) or (-)
• Morphology of the bacteria (cocci, diplococci,
streptococci, rods or coccobacilli)
• Whether the organisms are intracellular
• Presence & number of pus cells
• Presence of yeast cells & epithelial cells
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• Purpose : to stain Mycobacterium species
• Mycobacterium species do not stain well by
the Gram technique
• Carbol fuchsin combined with phenol, binds
to the mycolic acid in the mycobacterial cell
wall

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Reagent
Carbol fuchsin primary dye
stain
Acid alcohol 3% decolorization, removes the red
dye except Mycobacterium 
acid fast bacilli (AFB)
Methylen Blue counterstain, stains the
0,3% background (contrast)

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 Designed for  bacteria whose cell walls contain
long-chain fatty (mycolic) acids. Mycolic acids
render the cells resistant to decolorization, even
with acid alcohol decolorizers.
 Mycobacteria are the most commonly
encountered acid-fast bacteria.
 Some degree of acid-fastness is a characteristic of
a few nonmycobacterial bacteria  Nocardia spp.
& coccidian parasites (Cryptosporidium spp.).
Procedure:
1. Fix smear by heat
2. Cover with carbol fuchsin, steam gently for 5
minutes over direct flame (or for 20 minutes over a
water bath). Do not permit slides to boil or dry out.
3. Wash with deionized water.
4. Decolorize in 3,0% acid-alcohol (95% ethanol and
3,0% hydrochloric acid) until only a faint pink color
remains.
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Procedure:
5. Wash with water
6. Counterstain for 1 minute with Loffler’s
methylene blue.
7. Wash with deionaized water and let dry.

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Ziehl-Neelsen (ZN) staining procedure
1 2

3 4 5

washing 3% acid alcohol (5’)

carbol fuchsin+heat  5’

6 7

washing

methylene Blue 0,3% (1’-2’) IUATLD, 2000

 100x

Reporting system for AFB

AFB seen Reporting System
No AFB seen. Examine at least 100 Negative / No AFB
fields seen
1-9 AFB / 100 field Report actual number
10-99 AFB / 100 field 1+
1-10 AFB / field. Examine at least 2+
50 fields
> 10 AFB / field. Examine at least 3+
20 fields
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Other staining for AFB
• Kinyoun Staining
- more concentrated carbolfuchsin ensure stain
penetration
- heating (-)
• Auramine-phenol fluorochrome staining
- fluorescence technique
- AFB fluoresce brightly against a dark background
- fluorescence microscope (40x objective)
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Methylene capsule of Bacillus anthracis, staining
blue leucocytes in faecal preparations
Wayson’s shows clearly the bipolar staining
bipolar morphology of bacteria (Yersinia
pestis)
Albert to stain metachromatic granules of C.
staining diphtheriae

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Giemsa for Chlamydia trachomatis inclusion
bodies, Borrelia species
Yersinia pestis, Histoplasma species,
internal bodies of Pneumocystis
jiroveci cysts, Klebsiella granulomatis,
Penicillium marneffei
Acridine Trichomonas vaginalis, yeast cells, and
orange clue cells in vaginal smears,
intracellular gonococci, meningococci,
and other bacteria (blood cultures)
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 Based on the composition of the fluorescent stain

reagents, fluorescent staining techniques may be

divided into two general categories: fluorochroming, in
which a fluorescent dye is used alone, and
immunofluorescence, in which fluorescent dyes have
been linked (conjugated) to specific antibodies.

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 In fluorochroming a direct chemical interaction occurs

between the fluorescent dye and a component of the

bacterial cell; this interaction is the same as occurs with the
stains used in light microscopy. The difference is that use of
a fluorescent dye enhances contrast and amplifies the
observer’s ability to detect stained cells tenfold greater
than would be observed by light microscopy.

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1. Cheesbrough M. District Laboratory Practice in
Tropical Countries: Part 2, 2nd ed. New York:
Cambridge University Press. 2006.
2. Brooks GF, Carroll KC, Butel JS, Morse SA, eds. Jawetz,
Melnick, & Adelberg’s Medical Microbiology, 24th ed.
New York: McGraw-Hill. 2007

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THANK YOU