[CLINICAL BACTERIOLOGY] MLS 2309

Agarose
MODULE 4.2: TRADITIONAL CULTIVATION
• Sulfated polymer made up of D-galactose, 3,6-anhydro-L-
galactose, and D-glucuronic acid
LEARNING OUTCOMES: • Most common solidifying agent
At the end of this module, the learner should have been be • Melts at ≥95° C
able to: • Resolidify below 50° C
1. Describe the different types of culture media and • Cooled to 55-60°C for distribution into petri dishes
their purpose
2. Identify the various inoculation techniques for
the proper isolation of microorganisms
3. Identify the growth requirements for each
particular group of bacteria
4. Differentiate the stages in the bacterial growth
curve.
TRADITIONAL CULTIVATION AND IDENTIFICATION
Primarily, 3 things are done in Clin. Lab. Bacteriology.

Classification of Culture Media According to COMPOSITION

CULTURE MEDIA
• Any material containing the necessary nutritional and
environmental requirements for bacterial growth
• Classified according to:
1. Physical state/consistency
2. Composition
3. Distribution/dispensing
4. Function/use
Classification of Culture Media According to PHYSICAL
STATE/CONSISTENCY
Tissue Culture Systems
for obligate intracellular parasites

1 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

First Immortal Human Cell Line (1951): HeLa • Examples:

The cell line was derived from cervical cancer cells taken from 1. NA/NB
involuntary donor Henrietta Lacks, who died of cancer that 2. TSA/TSB (trypticase soy)
year. 3. Thioglycollate broth
The cells were propagated by George Otto Gey without Lacks' Thioglycollate Broth
knowledge or permission and later commercialized. • Only medium capable of supporting growth of:
The cells traveled around the globe- even into space, on a 1. Aerobes (surface of medium)
satellite to determine whether human tissues could survive 2. Microaerophiles (subsurface)
zero gravity- and have been used for research into cancer, 3. Anaerobes (bottom)
AIDS, the effects of radiation and toxic substances, gene • Stored @RT in the dark
mapping, etc. • absorbs O2 if refrigerated
Classification of Culture Media According to
DISTRIBUTION/DISPENSING

Enriched Media
• Contains additional supplements (e.g. blood, vitamins, yeast
extracts) for fastidious bacteria üFastidious bacteria – needs
are relatively complex
• Examples:
1. Blood agar plate (BAP)
2. Chocolate agar plate (CAP)
Blood Agar Plate (BAP)
• 5% defibrinated blood
• Choices: sheep > horse >rabbit
Classification of Culture Media According to FUNCTION • Human blood not preferred because it contains inhibitors:
1. Supportive Media • Citrate – inhibits β hemolytic strep
2. Enriched Media • Dextrose – alter hemolytic pattern
3. Enrichment media Chocolate Agar Plate (CAP)
4. Selective media • Blood ∆ @ 80°C
5. Differential media • RBCs are lysed to release Xfactor (hemin) and V factor (NAD)
6. Selective and Differential Media needed by fastidious bacteria
7. Special media • For recovery of S. pneumoniae, Haemophilus and Neisseria
8. Transport media Enrichment Media
9. Biochemical testing media • Enhance the growth of particular organisms and suppress
Supportive Media growth of normal flora
• aka Simple/Basal/General Isolation culture media • Incubated for a certain period and then subcultured to
• For routine cultivation isolate the desired organism
• Supports growth of most nonfastidious bacteria • Examples:
• Doesn’t provide growth advantage to any organism 1. APW – for Vibrio

2 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

2. Selenite and Tetrathionate broths – for Salmonella and Special Media

Shigella • Used to isolate bacteria with specific growth requirements
3. BCYE – Legionella pneumophila • Examples:
Selective Media 1. Middlebrook 7H-10 agar – MTB
• Contain inhibitor(s) to all organisms except the organism 2. Fletcher medium – Leptospira
being sought 3. W medium – Brucella
• Examples: 4. Bordet-Gengou agar – B. pertussis
1. Lowenstein Jensen for MTB 5. Thayer Martin – Neisseria
2. • inhibitor: Malachite green 2. Moeller Tellurite for 6. MacBride – L. monocytogenes
C. diphtheriae • inhibitor: K. tellurite 7. Dieadonnes medium – V. cholera
Gram-Pos Inhibitors Transport Media
• Crystal/Gentian violet • For collecting, transporting and preserving microbiological
• Basic/Carbol fuchsin specimens
• Bile salts (Na deoxycholate) • Examples:
1. Amies – throat, vaginal, and wound samples
Gram-Neg Inhibitors
2. Cary-Blair – for Vibrio cholerae
• K tellurite 3. Stuart’s – for N. gonorrhoea and B. pertussis
• Na azide
For swarming of bacteria
• Alcohol
• Chloral hydrate
Differential Media
• Distinguish organisms by their differences in cultural
characteristics
• Example:
1. Blood agar – hemolytic patterns

Biochemical Testing Media

• Used to demonstrate physiological and chemical
characteristics of microorganisms
• Examples:
1. TSI
2. LIA

Selective and Differential Culture Media

1. EMB, McConkey, HEA, SSA – for gram-neg enteric bacilli
2. MSA – for Staphylococci
3. TCBS – for Vibrio
Hemolytic Patterns on Blood Agar
1. Alpha (α) – incomplete zone of hemolysis; green/brown
zone
2. Beta (β) – complete hemolysis; colorless zone
3. Gamma (γ) – non hemolytic
 Alpha-prime – inner alpha zone and outer zone of
beta hemolysis ; seen when plates are refrigerate QC for Culture Media
For commercially prepared culture media:
1. Exempt from testing if purchased from manufacturer who
follows NCCLS guidelines.

3 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

2. Include manufacturer's verification in QC log DILUTION STREAK TECHNIQUE

For culture media prepared in the lab: for isolation and semiquantitation of bacterial colonies.
1. 5% of each batch (maximum of 10) should be checked for A. .Actual plates show sparse, or 1+ bacterial growth that is
sterility. Incubate for 48 hours. limited to the first quadrant.
2. Check daily for moisture, sterility, color, hemolysis. B. Moderate, or 2+ bacterial growth that extends to the
3. Check with organisms expected to grow or give positive second quadrant.
reactions, and organisms not expected to grow or give C. Heavy, or 3+ to 4+ bacterial growth that extends to the
negative reactions. fourth quadrant.
INOCULATION OF SPECIMEN
• Sterile body fluids, pus, urine, and sputum are inoculated
directly into the selected media
• Specimens received on swabs can be inoculated directly
into the culture media
Inoculation Techniques
• Streaking is the most common manner of inoculation •
Placement of fluid specimens or swabs into broth or liquid
media
• Stabbing of the medium is usually performed with group A
streptococci to create anaerobiasis and promote subsurafce
hemolysis

QUANTITATIVE ISOLATION TECHNIQUE

A, Streaking pattern using a calibrated loop for enumeration
GENERAL PURPOSE ISOLATION STREAK of bacterial colonies grown from a liquid specimen such as
• Swab specimen is inoculated by gently “rolling the tip of the urine.
swab” onto the upper portion of the plate B, Actual plate shows well-isolated and dispersed bacterial
• Liquid specimens are dropped. colonies for enumeration obtained with the calibrated loop
• The inoculated area should be streaked by a sterile loop streaking technique.
afterwards

4 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

Manner of Inoculation Nitrogen

• Inoculating loop is sterilized and allowed to cool thoroughly • Major component of proteins and RNA/DNA
before use • Most microorganisms use ammonia (NH3) as a sole
• Inoculating loop should be flamed between different media nitrogen source
plates, except when specimen is collected with swabs Sulfur
• Bedside or direct inoculation is used for the isolation of • Component of many organic cell substance
pathogens that are sensitive to drying or being transported • Most microorganisms use sulfate (SO4 2- ) as sulfur source
• Inoculation should start from nonselective to selective • Reduce sulfate to H2S
plates Phosphorous
BACTERIAL GROWTH REQUIREMENTS • Phosphate (PO4 3- ) component of ATP, nucleic acids,
NUTRITIONAL REQUIREMENTS coenzymes
1. Carbon • Phosphate assimilated as free inorganic phosphate (Pi)
2. Nitrogen Minerals
3. Growth factors • Mg2+ and K+ essential for ribosomes
4. Sulfur • Ca2+ constituent of gram-pos CW
5. Phosphorous • Fe2+ part of coenzymes of cytochromes and peroxidises
6. Minerals Classification of Organisms according to O2 Requirement
ENVIRONMENTAL REQUIREMENTS Aerobes Grows in the presence of O2 Classification:
1. Oxygen 1. OBLIGATE AEROBES
2. Carbon dioxide • Grows only in the presence of O2
3. Temperature 2. FACULTATIVE ANAEROBES
4. pH • Aerobes that can grow in the absence of O2
5. Moisture 3. MICROAEROPHILES
6. Osmotic pressure • Grows best @ ↓ O2 tension
7. Ionic Strength (salt) Anaerobes Grows in the absence of O2 Classification:
Carbon 1. OBLIGATE ANAEROBES
• For synthesis of cellular components • Grows only in the absence of O2
• All bacteria that inhabit the human body fall into the 2. AEROTOLERANT ANAEROBES
heterotrophic group • Also FACULTATIVE AEROBES
CLASSIFICATION • Anaerobes that can grow in the presence of O2
AUTOTROPHS • Don’t grow well but survive in the presence of O2
aka Lithotrophs Oxygen is toxic to OBLIGATE ANAEROBES because they lack
• C from inorganic substance (CO2) superoxide dismutase and catalase.
HETEROTROPHS Oxygen Toxicity
•aka Organotrophs • End products of O2 (superoxide & H2O2) are toxic
• C from organic substance (CHO, CHON, lipids) • Superoxide dismutase and catalase converts toxic
•more common substance to non toxic forms
Growth Factors
• Organic substances essential for growth
• Usually provided in the culture medium
• Ex: vitamin B complex, amino acids, purines, pyrimidines,
fatty acids, pentoses
CLASSIFICATION ANAEROBIC JARS: GasPak Jar
• Organic substances essential for growth Used to culture obligate anaerobes Components:
• Usually provided in the culture medium 1. Hydrogen and CO 2
• Ex: vitamin B complex, amino acids, purines, pyrimidines, 2. Generator envelope
fatty acids, pentoses 3. Catalyst: Palladium
PROTOTROPHICS 4. Indicator: Resazurin or Methylene Blue
•Don’t require exogenous source Indicator
•Can synthesize their own  without O 2 = white
AUXOTROPHICS  with O 2 = pink or blue
•Require addition of GF for growth to culture media Quality Control
Check for anaerobiosis with methylene blue strip each use

5 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

Candle Jar
• Used to culture Capnophilic organisms
• 5% CO2 & 15% O2
• Not used to culture obligate anaerobes!
INCUBATORS
Can be regulated to mimic a cell’s natural environment: 1.
temperature of 35-37°C
2. relative humidity of about 95%
3. CO2 concentration of 3- 5%
Quality Control: check CO2 concentration and temperature
daily
Temperature
• Optimal temperature: temperature @ which an organism
THE GASPAK ANAEROBIC SYSTEM grows best
Principle: water added to the CO2 and H2 generator • Opt. temp. for most bacteria is 35 – 37°C
envelope and O2 catalyzed with H2 to H2O via the pellets, • Incubation at certain temp. used as enrichment procedure
anaerobiasis is achieved • Cold Enrichment Technique
CLASSIFICATION
PSYCROPHILIC
• -5 – 15°C
• Also Cryophiles, cold loving
PSYCHROTROPHS
• 20 – 30°C
• Most organisms
MESOPHILIC
• 30 – 37°C • Most organisms
THERMOPHILIC
• Heat loving, 50 – 60°C
• Hyperthermophiles: 50 – 125°C
pH
• Hydrogen ion concentration
Carbon Dioxide • Optimal pH for most pathogenic bacteria of 6.5 – 7.5
• Capnophilic organisms require increased CO2 (5 -10% CO2) • Lab. culture media usually pH 7.0 – 7.5
• Examples: CLASSIFICATION
a. Neisseria ACIDOPHILES
b. Brucella • pH 1.0 – 5.0
c. HACEK group • e.g., Lactobacillus
• Most aerobic and facultative aerobic bacteria needs 0.03% NEUTROPHILES
CO2 • pH 5.5 – 8 . 5
• Most
ALKALOPHILES
• pH 9.0 – 11.0
• e.g., Vibrio
Inorganic Salt
• Halophilic organisms Requires ↑ salt concentration
• Examples:
1. Vibrio
2. Staphylococcus

6 [BONILLA, ZYRA]
 [CLINICAL BACTERIOLOGY] MLS 2309

Lag Phase
Moisture
• Little or no multiplication
• Indispensable!
• Period of adaptation
• solvent for food
• Bacteria very active metabolically
• forms major portion of protoplasm
Log Phase
• Humidophilic organisms require ↑ moisture
• Maximal rate of cell division
• Vital for bacterial growth and susceptibility testing
• Most sensitive to antimicrobials
Pressure
• Bacteria most active metabolically
• Barophiles grow rapidly in high-pressure environment (600-
Stationary Phase
1100 atm pressure)
• Rate of cell reproduction equals cell death
• Examples: Photobacterium, Shewanella, Colwellia
• Growth ceases due to:
Incubation Conditions
a. nutrients exhaustion
b. waste accumulation
c. change in pH
Decline Phase
• Complete cessation of multiplication

Incubation Conditions
• For most bacteria, ideal incubation temp. is 35ºC
• Aerobic bacterial cultures should be examined after 18-24
hours of incubation, for anaerobic cultures 48 hours Methods for Measuring Bacterial Growth
• Most routine bacterial cultures are held for 48-72 hours
• Cultures for anaerobes and broth cultures may be held for
5-7 days
PHASES OF BACTERIAL GROWTH
1. LAG PHASE
Phase of Rejuvenescence or Phase of Physiologic Youth
2. LOG PHASE
Logarithmic/Exponential phase
3. STATIONARY PHASE
Phase of Equilibrium or Plateau phase
4. DECLINE PHASE
Death phase

7 [BONILLA, ZYRA]