MEDIA PREPARATION AND METHODS OF INOCULATION

GOAL OF MICROBIOLOGY:

 Identifying and isolating particular microorganisms for diagnosis of certain diseases: identify what
causes the infection of the patient.
 (identifying and isolating particular microorganisms made possible by utilizing effective and appropriate
culture media for their growth, transport, and storage)- in order to enhance growth and survival
 -rely on their ability to grow and survive outside the host that’s why we’re using culture media;
technically letting them grow in culture media to identify what will grow from a patient’s sample
Example:

Female patient; lower back pain, painful urinating; initial diagnosis of doctor is UTI. In order to confirm this
diagnosis, the patient will submit urine sample in the lab then the urine sample we have to culture it; and
identify what significant microorganism grew (e.g. E. coli) the presence of E. coli relevant number, their
colony; is indication of UTI.

CHARACTERISTICS OF CULTURE MEDIA

 Microorganisms are grown in the laboratory using various types of culture media
 However, the culture media it may depend on the type of bacteria to be isolated/ identified
 Culture media is composed of mixture of nutrients
 These different nutrients they may be incorporated to the culture media; usually the nutrients that
bacteria needed are supposed to be high in sugar and protein
 Ex: if we want to preserved glucose we need to prevent bacterial growth, bacteria wants glucose, they
highly consumed glucose; that is why glucose/ any forms of sugars is incorporated in various type of
culture media.

 These various nutrients they’re being added to our culture media for specific bacterial growth
requirements and aside form sugar and protein it may be added with – -carbon, nitrogen, sulfur,
phosphorous, hydrogen, oxygen, buffers (pH buffers), pH indicators (importance in culture
media)
 usually added for differentiation (of microorganisms) purposes based on their biochemical reaction
 Mac Conkey-ph indicators to see what bacteria grow in it; is it non-bacteriaceae/ bacteriaceae
 We can also incorporate inhibitory agents: Dyes, antibiotics, etc.
 These inhibitory agents they may be incorporated in the culture media; we want to inhibit other
growth of other microorganism; in order to facilitate the isolation of the desired microorganism

Ex. Culture media for salmonella and shigella, hektoen enteric agar

 Like dyes: so that selectively salmonella and shigella will only grow in a culture media
 In order to facilitate their isolation while suppressing the growth of other microorganisms which are
present in the sample.

NOTES TO REMEMBER:

-Optimum growth could only be achieved using

combined proper use of:

*culture media (proper use and content)

*growth temperature (proper use)

*Nutrient supplementation
CULTURE AND CULTURE MEDIA

CULTURE
-growth of microorganism on culture medium (colonies that grow)
 PURE CULTURE
- contains only 1 genus (pure culture of staphylococcus)
 MIXED CULTURE
- contains more than 2 genus and species; contain mixture of staphylococcus and streptococci
 STOCK CULTURE
- used for academic and research purposes; industrial purposes
CULTURE MEDIA
 a liquid, semi-solid, or solid preparation utilized to observe growth
pattern of microorganism as well as for transport and storage
Culture
CLASSIFICATION OF CULTURE MEDIUM

a. According to consistency (liquid, semi-solid/ solid)

1. Liquid medium
 0% agar
 allows growth of aerobes, anaerobes, and facultative aerobes

Brain heart infusion broth; Tryptic Soy Broth Thioglycollate

(Neisseria meningitidis, Streptococcus pyogenes)

2. Semi-solid medium
 contains 0.5-1% agar
 facilitates the motility of bacteria
 Ex. SIM (Sulfide Indole Motility)
Positive- turbidity; hazy after incubation (bacteria is motile)
Negative-straight; stays the same (non-motile)

3. Solid media + -
 contains 2-3% agar
 ex. TSIA, Mac Conkey, BAP (Blood Agar Plate), CAP
(Chocolate Agar plate)
BAP (Blood Agar Plate) CAP (Chocolate Agar plate)
 They both contain blood
 need to maintain and obtain the RBCs  In CAP the blood is hemolyzed
intact (need by Hemolyzing the RBCs
streptococci group) intentionally
PURPOSE:
We need the HEMIN
(homophilus)
NOTES TO REMEMBER:
The agar will melt if the temperature reaches 80˚C to 90˚C and solidify at 40˚C to 50˚C
The cooling temperature for distribution of agar into plates is 55˚C to 60˚C

b. According to composition
1. Synthetic medium
 All substances are known to the user
 used for research purposes
2. Non-synthetic/Complex medium
 composed of some unknown substances (peptones, yeast, meat, etc.)
 useful for the isolation of bacteria
 Ex: Nutrient broth, TSB, MAC
3. Tissue Culture medium
 used to isolate obligate intracellular bacteria (Rickettsia, Chlamydia)
bacteria that need living cell
 Ex. W138, HELA 229 cells, McCoy cells
used for cultivation of chlamydia spp.

c. According to how the medium is dispensed (plated, tube, slant-butt)

PLATED TUBED

Disperse in Petridish Slant Butt Slant with butt

d. According to their specific use

1. Simple Media/General purpose media/ Supportive media

 routinely used in the laboratory
 without added supplement
 support the growth of most non-fastidious bacteria
 ”walang arte”; can put it in simple media
 Ex. Nutrient Agar, Nutrient broth, TSB
2. Enrichment Media
 -used to propagate the growth of certain group of organisms
 -contains specific nutrients
o -adding specific nutrients in order to promote the growth of our desired micro organism
 Ex. Alkaline Peptone water, Selenite F broth, Thioglycollate, Gram negative broth Ex:
o Alkaline Peptone water used in isolate of vibrio microorganism (wants alkaline environment);
high in pH
3. Enriched Media
 with added supplements necessary for the growth of fastidious organisms
 Ex. Neisseria (Thayer-Martin Agar, Martin Lewis Agar)-added antibiotics
 solid-type medium
 EX. BAP, CAP

4. Differential Media
 media that allows visualization of metabolic differences between group of species of bacteria
 o differentiating groups of microorganism according to their biochemical reaction/ metabolic
difference
 Example: MacConkey, BAP, EMB, HEA
MacConkey

 differentiates lactose fermenters (enterobacteriaceae) from non-lactose fermenters (gram positive microorganism)

BAP (Blood Agar Plate)

 differentiate microorganism according based on hemolytic pattern (streptococci)

 can also be differential

 Beta-complete hemolysis (pyogenes)
 Alpha - incomplete
 Gamma-no hemolysis

pH indicators - incorporate in media in order to differentiate them

1. Phenol red
-TSIA, XLD (Xylose Lysine Deoxycholate agar), MSA, Christensen’s urea agar
2. Bromthymol Blue
-HEA, TCBS lactose fermenters (enterobacteriaceae) from non-lactose fermenters (gram positive microorganism)
differentiates
3. Neutral Red
-MAC, SSA
(gram positive microorganism)
4. Bromcresol purple
-LIA

5. Selective Media
 It is incorporated with antibiotics, dyes, or chemicals to inhibit the growth of other
organisms while promoting the growth of the desired organism
Ex. Stool sample (many different bacteria), patient is suspected with salmonella infection (can use
SSA)
 Examples
XLD-salmonella and shigella
GBA- Streptococci
MSA-Staphylococcus (very tolerant to high salt concentration)
Bacitracin BAP- Haemophilus
PEA- gram (+) organisms
Columbia CAN with blood - gram (+)
TMA- Neisseria gonorrheae
GN Broth- Salmonella and Sgigella

Inhibitory agents
 could be in the form of dye
Crystal violet, Inhibitory to gram (+)
Basic Fuschin with the presence of these dyes in culture media, they will inhibit gram (+)
Bile salt microorganism
Potassium Tellurite, Inhibotory to gram (-)
Sodium azide could inhibit gram (-) microorganism
 Alcohol chloral hydrate inhibitory to swarming phenomenon
Special Media
 To isolate bacteria with specific growth requirements
Ex.
 Lowenstein Jensen (LJ medium)-mycobacteria (TB)
 Thiosulfate citrate bile salt sucrose (TCBS)-isolation of vibrio spp

METHODS OF INOCULATION

What is Isolation streaking?

To study the properties of a given organism, it is necessary to handle it in pure culture free of all other
types of organisms. To do this, a single cell must be isolated from all other cells and cultivated in such a
manner that its collective progeny also remains isolated. Several methods are available

 Observe septic technique

 Flame sterilized inoculating loop
 Smear thoroughly our inoculum
 The purpose is we want to really
 Isolate microorganisms that we
want to identify

 NON-CALIBRATED NICHROME LOOPs

- size 2,3,4,5 mm
 PLASTIC (disposabal)
 INCOCULATING NEEDLE
- use for tube culture media (stab, and streak)

METHODS OF ISOLATION STREAKING

• Pour plate method

- A suspension of cells is
mixed with melted agar
at 50˚C and poured into a
petri dish -when the agar
solidifies, the cells are
immobilized in the agar
and grow into colonies.
 • Streak plate method
 as the streaking continues, fewer and fewer
cells are left on the loop, and finally the
loop may deposit single cells on the agar
 the plate in incubated, and any well-
isolated colony is the removed,
resuspended in water, and again streaked
on agar.

• Spread plate technique

o -a small volume of dilute microbial
suspension containing 30-300 cells is
transferred to the center of an agar plate
and spread evenly over the surface with a
sterile bent- glass rod.
o -the dispersed cells develop into
isolated colonies

TECHNIQUES OF INOCULATION
 Liquid
- The sample on the swab we can just
put it in the liquid broth and cut the
excess stick of swab and cover.

- using inoculating needle, you can just touch it on the tube

 Semi-solid
-
SIM- stab inoculating needle on the agar and incubate it. The sample on the swab we can just put it in
the liquid broth and cut the excess stick of swab and cover.
- using inoculating needle, you can just touch it on the tube
 Slant and slant/butt
 Plated medium
- Uninterrupted streak method
- interrupted streak method

o radial streak method

o overlap streak method
o Purpose: For antimicrobial susceptibility testing
TEMPERATURE REQUIREMENT

 Cultivation of most medically important bacteria is done using incubators with temperatures
maintained in the 35˚C to 37˚C range.
 Most of the pathogenic bacteria they grow in this temperature - 35˚C to 37˚C
 For fungi 28˚C

OXYGEN AND CARBON DIOXIDE AVAILABILITY

• Aerobic bacteria
- use oxygen as a terminal electron acceptor and grow well in room air
• Facultatively anaerobic
- being able to grow in the presence or absence of oxygen
• Microaerophilic
- needs low level of oxygen
• Anaerobic bacteria
- are unable to use oxygen as an electron acceptor, but some aerotolerant strains will still grow
slowly and poorly in the presence of oxygen.

INCUBATION CONDITIONS

Aerobes 21% O2 + 0.03% CO2

Anaerobes 0% O2, 5-10% CO2 + H2, 80-90% N2
Capnophiles 5-10% CO2 + 15% O2 (candle jar)
Microaerophiles 5-10% O2 + 8-10% CO2

MOISTURE

 Less water is available for essential bacterial metabolic pathways (they don’t grow without moisture)
 with a loss of water there is a relative increase in the solute concentration of the media.
 Increased atmospheric humidity enhances the growth of certain bacterial species.

METHODS FOR PROVIDING OPTIMUM INCUBATION CONDITIONS

 Incubators are the primary laboratory devices used to provide the environmental conditions required
for cultivating microorganisms.
 Once inoculated with patient specimens, most media are placed in incubators with temperatures
maintained between 35˚ and 37˚ C and humidified atmospheres that contain 3% to 5% CO2