Central Philippine University | College of Medical Laboratory Science KEY CONCEPTS

MEDICAL LABORATORY SCIENCE INTERNSHIP

I. MICROSCOPY
MICROBIOLOGY A. General Information
MODULE 1. Microscopy is the use of a microscope to magnify objects too small
3 LABORATORY TESTING OF for the naked eye.
BACTERIAL INFECTIONS 2. Microscopic Examination
a. Living State: Direct wet mount, hanging drop method
b. Fixed State: Physical fixation (e.g. heat), chemical fixation (e.g.
95% methanol)
MODULE OUTCOMES B. Bright-Field (Light) Microscopy
At the end of this module, the learner should have been able to: 1. Object appear dark against a light background
1. describe comprehensively the roles of a medical technologist in a 2. Key Components
microbiology laboratory. a. Magnification – objective lens and ocular lens
2. discuss accurately the different cultivation, isolation, and identification b. Resolution – detail maintained
techniques employed in the testing of bacterial infections. c. Contrast – make objects stand out from background; achieved
3. differentiate properly the various methods of antimicrobial susceptibility by staining
testing. 3. Essential Parts of a Microscope
4. identify correctly the QA/QC parameters to ensure the reliability of a. Lens System: Oculars, Objectives, Coarse- and fine-adjustment
bacteriology procedures and results. knobs
b. Illumination System: Light source, Condenser, Field and iris
INTRODUCTION diaphragm
c. Body: Base, Body tube, Nosepiece
Primarily, three things are done in clinical laboratory bacteriology to test C. Phase Contrast Microscopy
bacterial infections – culture, identification, and antimicrobial susceptibility 1. Enhances visualization of elements with low refractive indices
testing. The bacteriology laboratory plays an essential role in patient care 2. Not commonly used
by providing the cause of infection and the effective antimicrobial to be 3. Used to ID fungi
used by physicians. Providing accurate results is not only needed but rapid 4. Eliminates fixing or staining living cells (for observation of viable
diagnosis of pathogens is also important for initiating effective antibiotic organisms)
administration and improving the outcomes of treatment. D. Fluorescent Microscopy
1. Visualization of naturally fluorescent substances or those stained
with a fluorochrome
2. Fluorescence is the property by which some atoms absorb light at
a particular wavelength and subsequently emit a light of a longer
wavelength
3. Mechanism

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 1

 a. Excitation filter selects the wavelength of light from the light II. BACTERIAL STAINING
source A. General Information
b. Fluorescent substances absorb the energy and emit a longer 1. Staining is the process of artificially coloring microorganisms with
wavelength of light dyes
c. Emission filter selects a specific wavelength of emitted light 2. Objectives of Staining
from the specimen a. To determine the morphology of bacteria
4. Fluorochroming involves nonspecific staining of any bacterial cell b. To differentiate groups of bacteria
with a fluorescent dye. c. To identify organisms with special structure
5. Fluorochroming Methods B. Staining Techniques
a. Acridine Orange: stains all nucleic acid bright orange; confirm 1. Simple Stains
presence of bacteria in blood culture, stain for cell wall-deficient a. Utilizes only one dye
bacteria b. Color forms and shapes of the cell
b. Auramine-Rhodamine: stains mycolic acid (Mycobacteria cell c. Ex: Crystal violet, methylene blue
wall) bright yellow or orange 2. Differential Stains
c. Calcofluor White: stains fungi cell wall blue-white a. More than one dye added in several steps
6. Immunofluorescence uses antibodies labeled with fluorescent dye b. Color components of the cell
(i.e., a conjugate) to specifically stain a particular bacterial species. c. Ex: Gram stain, Acid-fast stain
E. Dark-Field Microscopy 3. Negative Staining
1. Specimen appears light against a black background a. Stains the background rather than the bacteria
2. Visualization of specimens not viewed with a bright-field b. Demonstrates capsules
microscope c. Ex: India ink
3. Used to detect Spirochettes (e.g. Treponema pallidum) C. Ionizable Dyes Used in Staining Bacteria
F. Electron Microscopy 1. Basic Dyes
1. Magnification of 100,000× or more a. Cationic dyes with positively charged groups
2. Uses electrons instead of light and, instead of lenses, the electrons b. Adhere to negatively charged molecules like nucleic acids and
are focused by electromagnetic fields and form an image proteins
3. Fixative: glutaraldehyde or osmium tetroxide c. Ex: methylene blue, basic fuchsin, crystal violet, safranin, and
4. Dehydrating agent: alcohol or acetone malachite green
5. Stain: lead citrate and uranyl acetate 2. Acidic Dyes
6. Two General Types a. Anionic dyes with negatively charged groups (carboxyls and
d. Transmission Electron Microscope (TEM) phenolic)
i. Passes electron beam through objects b. Binds to positively charged cell structures
ii. Visualization of internal structures c. Ex: eosin, rose bengal, and acid fuchsin
e. Scanning Electron Microscope (SEM) D. Smear Preparation
i. Electron beam scans surface of objects 1. Patient specimens
ii. three-dimensional views of surface structures a. Dropped using pipette (liquid) or rolled (swab) onto slide
2. Culture

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 2

 a. Solid medium: transfer to slide using needle, emulsify with Stearn and Stearn Low isoelectric High isoelectric
saline or water; rubbed to slide using wooden applicator stick point point
b. Liquid medium: aspirate sample and apply to slide Magnesium RNA Increased MgRNA Decreased MgRNA
E. Gram Stain Theory
1. Divides bacteria into: Techoic Acid Increased Techoic Lower Techoic Acid
a. Gram Positive Content Acid
i. Violet, purple or deep blue Lipid Content Decreased lipid Increased lipid
ii. Takes up the basic dye, crystal violet content content
iii. Thick peptidoglycan; ↑ techoic and teichuronic acids that 6. Reasons why Gram-Pos becomes Gram-Neg
forms a complex with CV and I2 a. Removal of MgRNA by precipitation with bile salts
b. Gram Negative b. Autolysis, aging and temperature of incubation
i. Red or pink c. Acid solution of Gram’s Iodine
ii. Don’t take up the primary stain d. Overdecolorization
iii. Primary stain washed out by the decolorizer 7. Reasons why Gram-Neg becomes Gram-Pos
iv. Takes up the counterstain a. Incomplete decolorization
v. Thin peptidoglycan; ↑ lipid b. Thick smear
2. General Rule 8. Non-stain Systems to Determine Gram Stain Reactions
c. All cocci are Gram-positive except Neisseria, Veilonella & a. LANA (L-alanine-4-nitroanilide): reagent impregnated in cotton
Branhamella swabs which turns yellow when touched to gram-neg bacteria
d. All bacilli are Gram-negative except “MCC-BLELA-PEMB” b. 3% KOH test: Stringlike material identifies gram-neg bacteria
(Mycobacteria, Clostridia, Corynebacteria, Bacillus, Listeria, F. Acid-Fast Stains
Erysipelothrix, Lactobacillus, Actinomyces, Propionibacterium, 1. Principle: Some bacteria’s cell walls contain mycolic acids. Mycolic
Eubacterium, Mobiluncus, Bifidobacterium) acids render the cells resistant to decolorization.
e. All spirochettes are Gram-negative 2. Most commonly encountered acid-fast bacteria are Mycobacteria
3. Reagents 3. Methods:
Gram-Positive Gram-Negative a. Ziehl-Neelsen
1° stain Crystal Violet Violet Violet b. Kinyoun
Mordant Gram’s Iodine Violet Violet G. Special Staining Methods
Decolorizer 95% Alcohol Violet Colorless Cellular Structure Staining Technique
2° stain Safranin Violet/ purple Red/pink Cell wall Dyar
4. Quality Control: Control slides weekly. Use Escherichia coli and Capsule India ink, Nigrosin
Staphylococcus aureus. Hiss’, Gin’s, Anthony’s, Welch’s, Tyler’s,
5. Gram Staining Reaction Theories Muirs, Wadsworth, MacNeal, Lawson
Gram-Positive Gram-Negative Flagella Gray, Leifson, Fisher and Conn, Casares-
Theory
Bacteria Bacteria Gil’s, Loeffler’s, Van Ermengens
Benian Less permeable to Permeable to Metachromatic Neisser, Albert
decolorizing agent decolorizing agent granules

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 3

 Endospore Dorner, Wirtz and Conklin, Schaefer-Fulton b. Exact composition of some/all substance is not known
DNA Feulgen (peptones, meat, yeast extracts)
c. Ex: meat extract broth, NB, TSB, MacConkey
3. Tissue
III. TRADITIONAL CULTIVATION a. Contains living tissues
A. Culture Media b. Used for obligate intracellular bacteria
1. Any material containing the necessary nutritional and c. Examples: Chick embryo (viruses and Rickettsia) and McCoys
environmental requirements for bacterial growth (Chlamydia)
2. Classified according to: d. Tissue Culture Systems for obligate intracellular parasites
a. Physical state/consistency Example Source
b. Composition 1. VERO cells African Green Monkey Kidney
c. Distribution/dispensing 2. A549 cells Lung Carcinoma
d. Function/use 3. HeLa Cervical Carcinoma
B. Classification of culture media according to Physical State 4. HEP-2 cells Laryngeal Carcinoma
1. Liquid 5. McCoys Mouse Cell Lines
a. No solidifying agent/agar E. Classification of culture media according to Distribution/Dispensing
b. Ex: broth, infusion, milk 1. Tubed Media
2. Semisolid a. In sterile test tubes
c. Contains 0.5-1.5% agar b. Weigh > dissolve > dispense > sterilize
d. Ex: motility medium, transport medium c. Types of Tubed Media: Broth, slant, butt and butt/slant
3. Solid 2. Plated Media
a. Liquefiable: contains 2-3% agar (Ex: BAP, CAP, TSI) a. In petri dishes
b. Non-Liquefiable (Ex: chopped meat, rice grain) b. Sterilize 1st before dispensing
C. Agarose c. Weigh > dissolve > sterilize > dispense
1. Sulfated polymer made up of D-galactose, 3,6-anhydro-L- F. Classification of culture media according to Function
galactose, and D-glucuronic acid 1. Supportive Media
2. Most common solidifying agent a. aka Simple/Basal/General Isolation culture media
3. Melts at ≥95° C and resolidify below 50° C b. For routine cultivation
4. Cooled to 55-60°C for distribution into petri dishes c. Supports growth of most nonfastidious bacteria
D. Classification of culture media according to Composition d. Doesn’t provide growth advantage to any organism
1. Synthetic e. Examples:
a. Also defined medium i. NA/NB
b. Exact composition known ii. TSA/TSB (trypticase soy)
c. Ex: BG-11 medium iii. Thioglycollate broth: only medium capable of supporting
2. Nonsynthetic growth of aerobes (surface of medium), microaerophiles
a. Also complex medium (sub-surface), and anaerobes (bottom); stored @RT in the
dark; absorbs O2 if refrigerated

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 4

2. Enriched Media 5. Differential Media
a. Contains additional supplements (e.g. blood, vitamins, yeast a. Distinguish organisms by their differences in cultural
extracts) for fastidious bacteria characteristics
b. Fastidious bacteria – needs are relatively complex b. Example: Blood agar (hemolytic patterns)
c. Blood agar plate (BAP) i. Alpha (α) – incomplete zone of hemolysis; green/brown
i. 5% defibrinated blood zone
ii. Choices: sheep > horse >rabbit ii. Beta (β) – complete hemolysis; colorless zone
iii. Human blood not preferred because it contains inhibitors: iii. Gamma (γ) – non hemolytic
iv. Citrate – inhibits β hemolytic strep iv. Alpha-prime – inner alpha zone and outer zone of beta
v. Dextrose – alter hemolytic pattern hemolysis ; seen when plates are refrigerated
d. Chocolate agar plate (CAP) c. Selective and Differential Culture Media
i. Blood ∆ @ 80°C i. EMB, McConkey, HEA, SSA – for gram-neg enteric bacilli
ii. RBCs are lysed to release X-factor (hemin) and V factor ii. MSA – for Staphylococci
(NAD) needed by fastidious bacteria iii. TCBS – for Vibrio
iii. For recovery of S. pneumoniae, Haemophilus and Neisseria 6. Special Media
3. Enrichment Media a. Used to isolate bacteria with specific growth requirements
a. Enhance the growth of particular organisms and suppress b. Examples:
growth of normal flora i. Middlebrook 7H-10 agar – MTB
b. Incubated for a certain period and then subcultured to isolate ii. Fletcher medium – Leptospira
the desired organism iii. W medium – Brucella
c. Examples: iv. Bordet-Gengou agar – B. pertussis
i. APW – for Vibrio v. Thayer Martin – Neisseria
ii. Selenite and Tetrathionate broths – for Salmonella and vi. MacBride – L. monocytogenes
Shigella vii. Dieadonnes medium – V. cholerae
iii. BCYE – Legionella pneumophila 7. Transport Media
4. Selective Media a. For collecting, transporting and preserving microbiological
a. Contain inhibitor(s) to all organisms except the organism being specimens
sought b. Examples:
i. Gram-Pos Inhibitors: Crystal/Gentian violet, Basic/Carbol i. Amies – throat, vaginal, and wound samples
fuchsin, Bile salts (Na deoxycholate) ii. Cary-Blair – for Vibrio cholera
ii. Gram-Neg Inhibitors: K tellurite, Na azide iii. Stuart’s – for N. gonorrhoeae and B. pertussis
iii. For swarming of bacteria: Alcohol, Chloral hydrate 8. Biochemical Testing Media
b. Examples: a. Used to demonstrate physiological and chemical characteristics
i. Lowenstein Jensen for MTB: inhibitor is Malachite green of microorganisms
ii. Moeller Tellurite for C. diphtheria: inhibitor is K. tellurite b. Examples:
i. TSI
ii. LIA

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 5

 Specimen Collection and Processing CHAPTER 6 121

For legend see opposite page.

B

D
minced with sterile scissors and forceps into small pieces suit- mated based on the extent of growth beyond the original area
able for culture. of inoculum. Growth in the first quadrant can be graded as
1+, or light growth; growth in the second or third quadrant
Isolation Techniques can be graded as 2+ to 3+, or moderate; and growth in the
Specimens can be inoculated to agar plates by using a gen- third or fourth quadrant can be considered 4+, or heavy
IV. INOCULATION OF SPECIMEN eral-purpose isolation streak to yield a semiquantitative esti- growth. Some specimensc. require
Heavy, or 3+technique
a quantitative to 4+:tobacterial growth that extends to the fourth
A. Inoculation Techniques mate of growth. The specimen is applied by rolling the swab quadrant.
determine the number of bacteria present. Urine specimens
or placing a drop of liquid specimen onto a small area at are inoculated using a quantitative isolation. Plates are inocu-
1. Streaking is the most common manner the edge ofofthe inoculation

plate
Agar

Flame
plate. Broth media can be inoculated by lated using a calibrated loop to deliver a specified volume. The 92 PART II General Principles in Clinical Microbiology
2. Placement of fluid specimens or swabs into broth
placing a few drops or liquid
of the liquid specimen media
into the tube of urine is mixed well, and the calibrated loop (0.01 or 0.001 mL)
broth or placing the swab into the broth. The inoculating is inserted into the urine and transferred to the culture media
3. Sterile body fluids, pus, urine, andloop sputum
is sterilized are inoculated
and allowed directlybefore
to cool thoroughly by making a single streak down the center of the plate.
into the selected media streaking the agar. The cooled loop is passed back and forth Without flaming, the loop is streaked back and forth through

Flame
Streak

Bacteriologic
through the inoculum in the first quadrant several times. The the original inoculum. Figure 6-3 provides a diagram of this
4. Specimens received on swabs can be inoculated directly intoof the

loop
pattern

A
first quadrant should be at least one quarter the plate, technique.

3
2
culture media and the streak lines should be close together. The plate is E. Quantitative
Incubation Isolation Technique
turned, and quadrant two is streaked by passing the loop
5. Stabbing of the medium is usually through the performed with a group
edge of the first quadrant few times and A then Once the medium 1. is Streaking pattern
inoculated, incubation using
conditions musta calibrated loop
streptococci to create anaerobiasis and promote subsurafce and
streaking the rest of the area. The plate is turned again, for enumeration
be considered. This includes both temperature and of bacterial colonies
environ-
the loop is passed through the edge of quadrant two a few mental atmosphere and is determined by the type of specimen Liquid
hemolysis times and into the rest of the third quadrant, finally passing and the pathogens that grown from The
may be detected. a laboratory
liquid pro- specimen such as specimen
of inoculum

B. Manner of Inoculation the loop over the final area of the agar streaks the fourth urine.
cessing area should contain a chart or table stipulating where A B
quadrant. It may be necessary to flame the loop or turn the each medium should be placed for incubation. Most bacteria
1. Inoculating loop is sterilized and loop
allowed to cool
over in between thoroughly
quadrants, depending before
on the number cultures are incubated at 35° to 37°  C. Aerobes grow in
Figure 7-10 A, Streaking pattern using a calibrated loop for enumeration of bacterial colonies g
B, Actual plate shows well-isolated and dispersed bacterial colonies for enumeration ob
use and type of bacteria present in the specimen. Laboratory ambient air, whereas anaerobes cannot grow in the presence technique.
personnel must adjust their technique as necessary. When of oxygen and require an anaerobic atmosphere. Laboratories
2. Inoculating loop should be flamed more between
than one agar different
plate is used,media the loopplates,
is flamed in canV. BACTERIAL
achieve GROWTH
an anaerobic environment through theREQUIREMENTS
use of jars,
except when specimen is collected with swabs
between plates to prevent carryover of a possible contami- bags, or aA. General
chamber. Information
Some bacteria are capnophiles and require TABLE 7-2 Semi-Quantitation Grading Procedure for Bacterial
Isolates on Growth Media
success or failure o
dures often depend
nant from one plate to another. Figure 6-2 shows a diagram an increased concentration of CO2. This can be achieved by
3. Bedside or direct inoculation is used
of this for the isolation of pathogens
technique. a candle jar, a1.CO2 Nutritional
incubator, jar, orRequirements:
bag. Microaerophiles Carbon, Nitrogen, Growth factors,
NUMBER OF COLONIES VISIBLE INSulfur,
EACH QUADRANT
tions. Criteria frequ
growth include the
that are sensitive to drying or beingThetransported
general-purpose isolation streak is useful for most Phosphorous, and
grow with reduced oxygen and increased CO 2 andMinerals
can be Score
#1 (Initial
Quadrant) #2 #3 #4
• Colony size (us
described in rela
specimens. The relative number of organisms can be esti- isolated using jars or bags. The length of incubation varies for
4. Specimens collected at bedside are more susceptible to 2. Environmental Requirements:
individual organisms. Most routine bacterial cultures are held Oxygen,
1+
Carbon
Less than 10
dioxide, medium, large)
• Colony pigmenta
2+ Less than 10 Less than
contamination Temperature,
for 48 to 72 hours. Cultures for anaerobes andpH, Moisture,
broth cultures Osmotic pressure, Ionic Strength 10 (salt)
• Colony shape (in
of the colony [Fig
may be held for 5 to 7 days. Unusual organisms may require
5. Inoculation should start from nonselective to selective plates B. Carbon
special medium or conditions beyond the routine. It is helpful
3+ Greater than
10
Greater
than 10
Less than
10
• Colony surface a
dull, dry, transpa
• Changes in agar m
C. General Purpose Isolation Streak 1. For synthesis of cellular components 4+ Greater than
10
Greater
than 10
Greater
than 10
Greater
than 5 (e.g., hemolytic
Specimen is swabbed or dropped color of pH indic
1. Swab specimen is inoculated by by pipette near the edge of the agar 2. Classification Note: This is a general guideline. Individual laboratories may vary in the examples, see Fig
plate in the center of the first quadrant. Specimen is placed on plate methods used for quantitation. • Odor (certain bac
gently “rolling the tip of the swab” 1
1 witha.1:100Autotrophs
or 1:000 L loop be helpful in pre
Many of these cr
onto the upper portion of the plate a. aka Lithotrophs Colonies growing the adjectives and de
bacterial pathogens. In other words, the media selected different laboratori
2. Liquid specimens are dropped. 2 Cross-streaks
b. C from inorganic after 18-24 substance
hour
incubation
(CO2) for growth is a clue to the type of organism isolated (e.g., used, nearly every
growth on MacConkey agar indicates the organism is identification begin
3. The inoculated area should be b. Heterotrophs most likely a gram-negative bacillus). Yeast and some description of the co
gram-positive cocci are capable of limited growth on Although careful
streaked by a sterile loop i. aka Organotrophs MacConkey agar. The incubation conditions that support is important, it is u
growth may also be a preliminary indicator of which colony morphology
afterwards 2 ii. C from organic substance (CHO, bacteria CHON, have been lipids)
isolated (e.g., aerobic versus anaero- ria of one species oft
bic bacteria). are nearly indisting
D. Dilution Streak Technique iii. more common (all bacteria that inhabit the
Relative Quantities human
of Each Colonybody
Type. Thefall
predomi- species. Additionally
nance of a bacterial isolate is often used as one of the morphologic diversi
1. for isolation and semiquantitation into the heterotrophic group) criteria, along with direct smear results, organism viru- acteristics may be typ
lence, and the body site from which the culture was strains of that specie
of bacterial colonies. C. Growth Factors obtained, for establishing the organism’s clinical signifi- Gram Stain and Sub
cance. Several methods are used for semiquantitation of nies during cultivati
a. Sparse or 1+: bacterial growth 1. Organic substances essential for growth bacterial quantities including many, moderate, few or a ing morphologies an
numerical designation (4+, 3+, 2+) based on the number for timely performa
that is limited to the first 2. Usually provided in the culture medium Colony Characteristics.
of colonies identified in each streak area (Table 7-2). The Gram stain
Noting key features of a bacterial cultured bacteria ar
quadrant. 4 3
3. Ex: vitamin B complex, amino acids, purines, pyrimidines,colony is important fatty
for any bacterial identification; decide which identi

b. Moderate or 2+: bacterial acids, pentoses

FIGURE 6-2 General-purpose isolation streak. FIGURE 6-3 Quantitative isolation technique.
growth that extends to the second quadrant.

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 6

 4. Classification c. Oxygen is toxic to obligate anaerobes because they lack
a. Prototrophics: don’t require exogenous source; can synthesize superoxide dismutase and catalase.
their own I. Carbon Dioxide
b. Auxotrophics: require addition of GF for growth to culture media 1. Capnophilic organisms require increased CO2 (5 -10% CO2)
D. Nitrogen 2. Examples: Neisseria, Brucella, and HACEK group
1. Major component of proteins and RNA/DNA 3. Most aerobic and facultative aerobic bacteria needs 0.03% CO2
2. Most microorganisms use ammonia (NH3) as a sole nitrogen J. Temperature
source 1. Optimal temperature:temperature @ which an organism grows best
E. Sulfur 2. Opt. temp. for most bacteria is 35 – 37°C
1. Component of many organic cell substances 3. Incubation at certain temp. used as enrichment procedure (e.g.,
2. Most microorganisms use sulfate (SO42-) as sulfur source Cold Enrichment Technique)
3. Reduce sulfate to H2S 4. Classification
F. Phosphorous a. Psycrophilic: also Cryophiles, cold loving; -5 – 15°C
1. Phosphate (PO43-) component of ATP, nucleic acids, coenzymes b. Psychrotrophs: 20 – 30°C
2. Phosphate assimilated as free inorganic phosphate (Pi) c. Mesophilic: 30 – 37°C; most organisms
G. Minerals d. Thermophilic: heat loving, 50 – 60°C
1. Mg2+ and K+ essential for ribosomes e. Hyperthermophiles: 50 – 125°C
2. Ca2+ constituent of gram-pos CW K. pH
3. Fe2+ part of coenzymes of cytochromes and peroxidases 1. Hydrogen ion concentration
H. Oxygen 2. Optimal pH for most pathogenic bacteria of 6.5 – 7.5
1. Classification of organisms according to O2 requirement 3. Lab. culture media usually pH 7.0 – 7.5
a. Aerobes: grows in the presence of O2 4. Classification
i. Obligate Aerobes: grow only in the presence of O2 a. Acidophiles: pH 1.0 – 5.0 (e.g., Lactobacillus)
ii. Facultative Anaerobes: aerobes that can grow in the b. Neutrophiles: pH 5.5 – 8.5 (most pathogenic bacteria)
absence of O2 c. Alkalophiles: pH 9.0 – 11.0 (e.g., Vibrio)
iii. Microaerophiles: grows best @ ↓ O2 tension L. Inorganic Salt
b. Anaerobes: grow in the absence of O2 1. Halophilic organisms require ↑ salt concentration
i. Obligate Anaerobes: grow only in the absence of O2 2. Examples: Vibrio and Staphylococcus
ii. Aerotolerant Anaerobes/Facultative Aerobes: anaerobes M. Moisture
that can grow in the presence of O2; don’t grow well but 1. Indispensable: solvent for food and forms major portion of
survive in the presence of O2 protoplasm
2. Oxygen Toxicity 2. Humidophilic organisms require ↑ moisture
a. End products of O2 (superoxide & H2O2) are toxic 3. Vital for bacterial growth and susceptibility testing
b. Superoxide dismutase and catalase converts toxic substance to
non toxic forms
2 O2- + 2 H+ --Superoxide dismutase→ O2 + H2O2
2 H2O2 --Catalase→ 2 H2O + O2

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 7

VI. INCUBATION AND PHASES OF BACTERIAL GROWTH 2. Generation/Doubing time: average time required for an organism to
A. General Information double its number (e.g., for E. coli = 20 minutes; MTB = 24 hours)
1. For most bacteria, ideal incubation temp. is 35ºC 3. Signs of bacterial growth:
2. Aerobic bacterial cultures should be examined after 18-24 hours of a. Turbid broth
incubation, for anaerobic cultures 48 hours b. Bacterial colonies on agar surface
3. Most routine bacterial cultures are held for 48-72 hours G. Phases of Bacterial Growth
4. Cultures for anaerobes and broth cultures may be held for 5-7 days 1. Lag Phase
B. Incubation Conditions a. Phase of Rejuvenescence or Phase of Physiologic Youth
Aerobes 21% O2 + 0.03% CO2 b. Little or no multiplication
Anaerobes 0% O2, 5-10% CO2 + 5-10% H2 + 80-90% N2 c. Period of adaptation
(anaerobe jars, bags, or chambers) d. Bacteria very active metabolically
Capnophiles 5-10% CO2 + 15% O2 (CO2 incubator/bag; 3% 2. Log Phase
CO2 in candle jars) a. Logarithmic/Exponential phase
Microaerophiles 5-10% O2 + 8-10% CO2 b. Maximal rate of cell division
C. Incubators c. Most sensitive to antimicrobials
1. Can be regulated to mimic a cell’s natural environment: d. Bacteria most active metabolically
a. temperature of 35-37°C 3. Stationary Phase
b. relative humidity of about 95% a. Phase of Equilibrium or Plateau phase
c. CO2 concentration of 3-5% b. Rate of cell reproduction equals cell death
2. QC: check CO2 concentration and temperature daily c. Growth ceases due to nutrients exhaustion, waste
D. Anaerobic Jars: GasPak Jar accumulation, and change in pH
1. Used to culture obligate anaerobes 4. Decline Phase
2. Components: a. Death phase Complete cessation of multiplication
a. Hydrogen and CO2 H. Methods for Measuring Bacterial Growth
b. Generator envelope Methods Application
c. Catalyst: Palladium Microscopic Count Enumeration of bacteria in milk and
d. Indicator: Resazurin or Methylene Blue vaccine
i. without O2 = white Plate Count Enumeration of bacteria in milk, food,
ii. with O2 = pink or blue water, and soil
3. QC: Check for anaerobiosis with methylene blue strip each use Membrane Or Molecular Enumeration of bacteria in food and
E. Candle Jar Filter water (including lakes and streams)
1. Used to culture Capnophilic organisms Turbidimetric Method Used to prepare standard inocula for
2. 5% CO2 and 15% O2 antimicrobial susceptibility testing
3. Not used to culture obligate anaerobes! Dry Weight For filamentous organisms (fungi)
F. Bacterial Growth Determination
1. Growth is the orderly increase of all chemical constituents of the Biochemical Activity Enumeration of bacteria in milk
cell.

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 8

VII. TRADITIONAL CULTIVATION AND IDENTIFICATION 6. Colony Surface Appearance
A. Cultural Characteristics a. Mucoid
1. Culture: growth of microorganisms obtained in a culture medium; i. Water-like, glistening, confluent
result of repeated divisions of one or few microorganisms ii. In organisms that form slimes or capsules
2. Colonies: visible macroscopic masses of growth on a solid culture iii. Ex: K. pneumoniae, S. pneumoniae, H. influenzae, etc
medium b. Smooth
3. Types of Culture i. Uniform texture and homogeneity
b. Pure (Axenic) Culture: made up of organisms belonging to one ii. Easily emulsified in NSS
specie iii. Virulent!
c. Mixed Culture: made up of organisms belonging to different iv. Ex: Salmonella, Shigella, E. coli, Serratia, Proteus
species c. Rough
d. Stock Culture: pure culture of organisms used as a supply for i. Granulated & rough
industry, research and student’s use ii. Hard to emulsify in NSS
4. Methods of obtaining pure culture iii. Usually indicates loss of virulence (enteric bacteria)
a. Streak plate method iv. Virulence (B. anthracis & MTB)
b. Pour plate method 7. Changes in Media resulting from bacterial growth
c. Use of selective media and media containing antibiotics a. Hemolytic pattern on BA
d. Animal inoculation test b. Changes in color of pH indicators
3. Colony Size c. Pitting of the agar surface
a. Usually measured in mm 8. Odor
b. Described in relative terms: Pinpoint, small, medium, large a. Never inhale directly from the plate!
4. Colony Pigmentation b. Determined when lid of culture plate is removed and odor
a. Pigment production is an inherent characteristic of a specific dissipates into environment
organism confined generally to the colony. c. Characteristic colony odor
b. Examples: red (Serratia marcescence), green (Psedomonas i. Pseudomonas aeruginosa – grape-like odor, sweet, fruity,
aeruginosa) taco chips, tortillas or corn chips
5. Colony Shape ii. Staphylococcus aureus – old sock
iii. Proteus mirabilis – putrid
iv. Haemophilus spp. – musty basement, “mousy”
v. Clostridium – horse stable
vi. Staphylococcus lugdenensis – sweet, hay-like
vii. Streptococcus anginosus – sweet, cake-like, caramel,
butterscotch
viii. Escherichia coli – floral/flowery
ix. Moraxella catarrhalis – pungent, semen-like
x. Eikenella corrodens – bleach
xi. Acinetobacter baumannii – dirty gym socks, gym locker

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 9

 xii. Proteus spp – rancid, rotten chicken soup Establishing Growth in various NaCl concentrations,
xiii. Actinomycetes, Streptomyces, Nocardia spp – rich, earthy, Inhibitor Profiles Susceptibility to optochin and solubility in
musty dirt scent after fresh rain bile, Bile Esculin test, and Ethanol survival
xiv. Gram-negative anaerobes – bad breath, morning breath 4. Commercial Identification Systems
9. Consistency a. Categorized as automated or manual
a. Determined by touching the colony with a sterile loop b. Detection strategies for end products:
b. May be brittle (splinters), creamy (butyrous), dry, waxy, and i. Colorimetry
sticky ii. Fluorescence
10. Terms used to describe colonial characteristics iii. Turbidity
a. Size: pinpoint, small, medium, large 5. Multitest Systems
b. Form: circular, filamentous, pinpoint, irregular a. API (Analytical Profile Index; bioMerieux Clinical Diagnostics)
c. Elevation: flat, convex (dome shaped), raised, umbilicate b. Enterotube II (Becton, Dickinson and Company)
(depressed center), umbonate (raised center) c. Micro-ID (Remel)
d. Margin: smooth or entire, rough, irregular, curled, filamentous d. Microscan (Siemens Healthcare Diagnostics, formerly Dade
e. Surface: dull, glistening, moist Behring)
f. Form of Margin: observe at the edge of the colony (swarming of e. Vitek (bioMerieux Clinical Diagnostics)
Proteus, star-like appearance of yeast) C. Immunodiagnosis
g. Consistency: mucoid, creamy, viscous, butyrous, brittle, sticky, 1. A diagnosis relating to immunology
dry, waxy 2. Diagnosis of disease based on antigen-antibody reactions
h. Density: translucent, transparent, opaque 3. Using antibodies to diagnose infectious diseases requires:
i. Color: white, golden, buff, red, gray a. demonstrating a fourfold rise in titer
B. Biochemical Tests b. distinguishing between IgG and IgM
1. Measures the amount or activity of a particular enzyme or protein 4. Precipitation
in a sample a. Double Immunodiffusion – detect antigens of systemic fungi
2. Determine the nutritional and metabolic capabilities of a bacterial b. Counterimmunoelectrophoresis (CIE) – no longer widely used
isolate c. Flocculation Tests – VDRL and RPR for Treponema
3. Nutritional Requirements and Metabolic Capabilities 5. Agglutination
Single Enzyme Catalase Test, Oxidase Test, Indole Test, a. Latex Agglutination – detect C. neoformans antigen in CSF, S.
Tests Urease Test, PYR test, and Hippurate agalactiae, C. difficile toxins, and rotavirus
Hydrolysis b. Coagglutination – ID of Lancefield groups streptococci, S.
pneumoniae, H. influenzae types A to F
Tests for Presence Oxidation and Fermentation Tests, Amino c. Liposome-Enhanced Latex Agglutination
of Metabolic Acid Degradation d. Hemagglutination Inhibition Assays – Rubella, influenza,
Pathways parainfluenza, etc.
Single Substrate Citrate test, Malonate Utilization Test, and 6. Immunofluorescent Assays
Utilization Acetate Utilization Test a. Either a direct (DFA) or indirect (IFA) technique

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 10

 b. Used to detect B. pertussis, L. pneumophila, Giardia, stranded DNA molecules in the primer extension step. New
Cryptosporidium, Trichomonas, HSV, Treponema, etc. DNA is then used for the next cycle.
7. Enzyme Immunoassay (EIA) d. Branched DNA Detection
a. Also enzyme-linked immunosorbent assay (ELISA) i. Capture probes attached to a surface anneal to target
b. Solid-Phase Immunoassay – for hepatitis B, HIV nucleic acid.
c. Membrane-Bound SPIA – detection of group A and group B ii. Target probes anneal to nucleic acid and to preamplifier
streptococci antigen probes.
8. Other Tests iii. Amplifier probes anneal to preamplifier probes, forming a
a. Neutralization Assays – Streptococcus pyogenes infection branched DNA (bDNA) structure.
b. Complement Fixation test (CF) – fungi, respiratory viruses, iv. Label probes (with bound alkaline phosphatase [AP])
arboviruses, Q fever. anneal to the bDNA structure. A large, amplified signal is
c. Radioimmunoassays (RIA) – Hepatitis antigens detected enzymatically when the AP substrate is added.
d. Western Blot – HIV confirmatory test 5. Strain Typing Techniques
D. Molecular Diagnostic Testing a. Used for epidemiologic purposes
1. Involve analysis of genes, rather than the analysis of gene products b. Categories
2. Basic molecular diagnostics in clinical microbiology: nucleic acid i. Nonamplifiable methods: Southern blotting, plasmid profile
hybridization assays, amplification techniques, and strain typing analysis, restriction enzyme digestion of chromosomal
techniques DNA, PFGE, and MLEE
3. Nucleic Acid Hybridization Assays ii. Amplified methods: RAPD, Rep-PCR, and MLST
a. Detect nucleic acid targets with labeled probes
b. Techniques:
i. Solid support nucleic acid hybridization: Southern and VIII. ANTIMICROBIAL AGENTS
Northern blots and ISH A. General Information
ii. In-solution nucleic acid hybridization 1. Natural or synthetic substance
4. Amplification Techniques 2. Kills (bactericidal) or inhibits (bacteriostatic) growth of
a. Exponentially increase either the amount of target nucleic acid microorganisms
or the signal that binds to the target nucleic acid. 3. May be antibacterial/antibiotic, antiviral, antifungal, and/or
b. Include PCR and derivations of PCR (e.g., RT-PCR, multiplex antiparasitic
PCR, and nested PCR), NASBA, TMA, bDNA detection, hybrid 4. Classification of Antibiotics
capture, and cycling probe technology. a. Natural Drugs
c. Polymerase Chain Reaction (PCR) i. Produced by bacteria or fungi
i. Template DNA is denatured by heat to yield two single- ii. Examples
stranded DNA strands. Source Antibiotics
ii. Temperature is cooled and the two single-stranded primers Bacillus subtilis Bacitracin
anneal to the template DNA strands. Bacillus polymyxa Polymyxin
iii. Temperature is heated to 72° C, and DNA polymerase adds Cephalosporium Cephalosporins
nucleotides to the primers to synthesize new double- Micromonospora purpurea Gentamycin

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 11

 Penicillium notatum Penicillin Penicillins Amoxicillin, Ampicillin, Dicloxacillin,
Streptomyces erythraeus Erythromycin Indanyl, Carbenicillin, Methicillin, Nafcillin,
Streptomyces fradiae Neomycin Penicillin G, Penicillin V, Piperacillin,
Streptomyces nodosus Amphotericin B Ticarcillin
Streptomyces noursei Nystatin Cephalosporins 1st gen: cefadroxil, cefazolin, cephalexin
Streptomyces venezuelae Chloramphenicol 2nd gen: cefaclor, cefprozil, cefuroxime,
b. Semi-Synthetic Drugs cefoxitin
i. Modified natural drugs with added chemical groups 3rd gen: cefdinir, cefixime, cefotaxime,
ii. Ex: ampicillin, carbenicillin, and methicillin ceftazidime, ceftibuten, ceftizoxime,
c. Synthetic Drugs ceftriaxone
i. Chemically-produced drugs 4th gen: cefepime
ii. Ex: sulfonamides, trimethoprim, chloramphenicol, isoniazid, Carbapenems Ertapenem, Imipenem, Meropenem
ciprofloxacin, and dapsone Monobactams Aztreonam
B. Chemotherapeutic Criteria d. Beta-lactamase inhibitors
1. Narrow-spectrum antibiotics i. Given with a beta-lactam antibiotic to provide effective
a. Acting only on a single or a limited group of microorganisms treatment
b. Ex: Isoniazid is active against mycobacteria ii. Have structural similarity with the B-lactam antibiotics and
2. Extended-spectrum antibiotics function as subtsrate of B-lactamase, thus reducing harm on
a. Effective against gram-pos and also against a significant the B-lactam antibiotic
number of gram neg iii. Examples: Clavulanic acid, Sulbactam, and Tazobactam
b. Ex: Ampicillin is effective against gram-positive and some gram 2. Glycopeptides
negative bacteria a. Bind to precursors of cell wall synthesis
3. Broad-spectrum antibiotics b. e.g., Vancomycin, bacitracin, teicoplanin, daptomycin
a. Affect a wide variety of microbial species 3. Vancomycin
b. Ex: tetracycline and chloramphenicol a. Binds to D-Ala-D-Ala terminal of growing peptide chain
C. Classification of Antibacterial Agents by their Sites of Action b. Usually ineffective against gram-neg
1. Inhibitors of cell wall synthesis c. Used to treat methicillin-resistant Staph. aureus (MRSA)
2. Inhibitors of protein synthesis E. Inhibitors of Protein Synthesis
3. Inhibitors of nucleic acid function or synthesis Mechanism of Spectrum of
Antimicrobial Class
4. Inhibitors of cell membrane function Action Activity
5. Inhibitors of metabolism Aminoglycosides Bind to 30S Gram-pos and
D. Inhibitors of Cell Wall Synthesis (e.g. tobramycin, amikacin, ribosomal gram-neg;
1. Beta-Lactams netilmicin, streptomycin, subunit not anaerobic
a. Bind enzymes involved in peptidoglycan production (i.e., PBPs) kanamycin) bacteria
b. For Both gram-pos and gram-neg Macrolide-Lincosamide- Bind to 50S Gram-pos;
c. Beta-lactam Antibiotics Streptogramin (MLS) Group ribosomal not against most
(e.g. erythromycin, subunit gram-neg

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 12

 azithromycin, Metronidazole Uncertain; Most potent
clarithromycin, clindamycin) breakage of against anaerobic
Ketolides Bind to 50S Gram-pos cocci; DNA strands gram-negs
(e.g., telithromycin) ribosomal macrolide- Rifampin Inhibits RNA Gram-pos;
subunit resistant strains; synthesis by certain gram-neg
fastidious gram- binding DNA- (e.g., N.
negs (e.g., H. dependent, RNA meningitidis)
influenzae and M. polymerase
catarrhalis) G. Inhibitors of Other Metabolic Processes
Oxazolidinones Bind to 50S Gram-pos; those Antimicrobial Class Mechanism of Action Spectrum of Activity
(e.g., linezolid) ribosomal resistant to other Nitrofurantoin Uncertain; may have Gram-pos and gram-
subunit antimicrobial several bacterial neg; used only to
classes enzyme targets and treat UTI
Chloramphenicol Bind 50S Gram-pos and directly damage DNA
ribosomal gram-neg; Polymyxins Disruption of cell Gram-neg; poor
subunit toxic to bone (e.g., polymyxin B membrane against most gram-
marrow! and colistin) pos
Tetracyclines Bind 30S Gram-pos. and Sulfonamides Interfere with folic Gram-pos and gram-
(e.g., demeclocycline, ribosomal gram-neg; acid pathway by neg
doxycycline, micocycline, subunit intracellular binding the enzyme
tetracycline) bacterial dihydropteroate
pathogens (e.g., synthase
chlamydia) Trimethoprim Interferes with folic Gram-pos and gram-
Glycylglycines Bind to 30S Gram-pos and acid pathway by neg;
(e.g., tigecycline) ribosomal gram-neg; those binding the enzyme frequently combined
subunit resistant to dihydrofolate with a sulfonamide
tetracycline reductase
F. Inhibitors of DNA/RNA Synthesis
Mechanism of Spectrum of
Antimicrobial Class
Action Activity IX. ANTIMICROBIAL RESISTANCE
Fluoroquinolones Inhibit DNA Gram-pos and A. General Information
1st gen: nalidixic acid synthesis by gram-neg 1. Antimicrobial Resistance is the ability of a microbe to resist effects
2nd gen: ciprofloxacin, binding DNA of medication; Microbe sometimes referred to as “superbug” (WHO)
ofloxacin, norfloxacin gyrases 2. May be environmentally-mediated or microorganism-mediated
3rd gen: levofloxacin 3. Biologic resistance – organism becomes less susceptible
4th gen: moxifloxacin 4. Clinical resistance – susceptibility has been lost; drug is no longer
effective

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 13

 5. Environmentally Mediated Antimicrobial Resistance 2. Extended-Spectrum beta-lactamase (ESBL)
Factor Example a. B-lactamases that can hydrolyze penicillins, broad-spectrum
pH Activities of erythromycin and cephalosporins and monobactams (even against newly
aminoglycosides decrease with decreasing developed B-lactams)
pH, activity of tetracycline decreases with b. Indicator drugs: aztreonam, ceftriaxone, and cefotaxime among
increasing pH others
Atmosphere Diminished activity of aminoglycosides in c. Carbapenems are active against ESBL-producing strains
absence of O d. ESBL activity is inhibited by clavulinic acid
2
F. Target Site Modification
Cation Mg++ and Ca++ compete with aminoglycosides 1. Chromosomal Mutation: mutations in penicillin-binding proteins
concentrations for binding sites on cell surface (PBPs) confer resistance to β-lactams
Presence of Enterococci use thymine to circumvent 2. Enzymatic Target Site Alteration: monomethylation or dimethylation
metabolites sulfonamides and trimethoprim of the amino group in 50S subunit of ribosome results in reduced
6. Microorganism-mediated Antimicrobial Resistance affinity of macrolides.
a. Intrinsic (naturally coded): Impermeability, Biofilms, Efflux, G. Acquisition of New Targets
Enzymatic Inactivation 1. Acquire cellular targets with reduced affinity for the antibiotic
b. Acquired (mutation or gene acquisition): Efflux, Target Site 2. Methicillin-resistant S. aureus (MRSA)
Modification, Acquisition of New Targets, Enzymatic a. S. aureus resistant to beta-lactams (methicillin, oxacillin,
Inactivation penicillin, and amoxicillin
B. Impermeability b. mecA encodes penicillin-binding protein (PBP2A) with reduced
1. Inability to penetrate the cell wall of bacteria affinity for β-lactam antibiotics
2. Structure of CW relevant to resistance: H. Methods for Specific Resistance Mechanisms
a. Lipopolysaccharide (LPS): negatively charged LPS serves as 1. Phenotypic Methods
barrier to negatively charged antibiotics a. Beta-Lactamase Detection
b. Outer membrane proteins (Omps) called porins: permit influx of i. Chromogenic cephalosporinase test: When beta-lactam
nutrients (and antibiotics) and efflux of wastes ring of cephalosporin in the disk is hydrolyzed by the
C. Biofilms Innate mechanisms bacterial inoculum, a deep pink color is produced
1. Decreased penetration of antibiotics through biofilm matrix ii. Penicillin-based acidimetric and iodometric tests: detect
2. Decreased O2 and nutrient accompanied by altered metabolic production of penicilloic acid from hydrolysis of penicillin by
activity a β-lactamase
3. Formation of persister cells b. ESBLs Detection
D. Efflux i. Testing using “indicator” drugs: disk diffusion with
1. Efflux pumps are transporter proteins that removes toxic cefpodoxime, ceftazidime, cefotaxime, ceftriaxone,
substances from the cell aztreonam (decreasing order of sensitivity)
E. Enzymatic Inactivation ii. Confirmatory: use B-lactamse inhibitors (e.g. clavulanic
1. β-Lactamases hydrolyze β-lactam antibiotics using two acid); if activity of cefotaxime or ceftazidime or both is
mechanisms of action: metallo-based and serine-based restored, the resistance is due to ESBL.

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 14

 2. Genotypic Methods ii. Day 2
a. Molecular methods (e.g., mec gene for MRSA, van gene for Determine MIC (tube w/ lowest concentration of antibiotic
VRE) that shows no turbidity). Subculture non-turbid tubes to agar
plates (use 0.01 ml standard loop)
iii. Day 3
X. ANTIMICROBIAL SUSCEPTIBILITY TESTING Determine MBC (lowest concentration that kills 99.9%
A. General Information bacteria; plate with ≤0.1% growth)
1. aka Antimicrobial Sensitivity Testing 5. Broth Microdilution
2. Test performed on bacteria isolated from clinical specimens to a. uses microtiter trays; volume 0.05 to 0.1 mL
determine which antimicrobial agents might be effective in treating b. also Microbroth dilution method
infections caused by the bacteria c. NCCLS reference method
3. Conventional Susceptibility Testing Methods d. 5 × 105 CFU/mL (final concentration)
a. Dilution Tests e. Same principle as broth macrodilution except that it utilizes
i. Broth Dilution Method “microdilutions” and OD readings are measured
ii. Agar Dilution Method C. Agar Dilution
b. Diffusion Tests 1. Procedure:
i. Disk Agar Diffusion Tests a. Doubling dilution of antimicrobial is incorporated into a single
ii. Gradient Diffusion Tests agar plate (1 drug = 6 plates)
4. Minimum Inhibitory Concentration (MIC) b. Test bacteria (104 CFU) are “spot” inoculated onto each plate
a. Expressed in ug/mL using a multipronged replicating device
b. Lowest concentration of antibiotic that inhibits growth c. Incubate. Examine plate for growth.
5. Minimum Bactericidal Concentration (MBC) d. Determine MIC.
a. also Minimum Lethal Concentration (MLC) 2. Reference method for testing of anaerobes and N. gonorrhoeae
b. Lowest concentration of antibiotic that kills 99.9% of bacteria D. Disk Agar Diffusion Test
B. Broth Dilution Method 1. Also Kirby-Bauer Technique
1. Also tube dilution method 2. Disk impregnated with antibiotic applied onto agar streaked with
2. Serial dilution of antibiotic is prepared, and to each tube a standard organisms
amount of inoculum is added 3. Limited to rapidly growing aerobic and facultative anaerobic
3. Determines MIC (can be continued to determine MBC) bacteria
4. Broth Macrodilution 4. Kirby-Bauer Technique Procedure
a. uses test tubes; volume 1mL or greater a. Pick 4-5 pure isolated colonies. Subculture to broth (TSB) or
b. Procedure: NSS and incubate @ 35°C for 2-8 hours.
i. Day 1 b. Compare turbidity with 0.5 McFarland Standard
Add 1 ml of test bacteria (1 × 108 CFU/ml) to tubes i. Also BaSO4 Standard
containing 1 ml broth and concentration of antibiotic (mg/l). ii. 99.5mL 1% H2SO4 + 0.5mL 1.175% BaCl2
Final standard bacterial concentration of 5 × 105 CFU/mL in iii. Equivalent to 1.5 × 108 CFU/mL
each tube is achieved. iv. Adjust turbidity if necessary. Add NSS or add colonies.

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 15

c. Streak swab into plated media. Dip swab into suspension. 5. Sources of Error in the Kirby-Bauer Technique
Streak swab on the medium 3×, rotating plate at 60° after each Source Comment
application. Use of mixed culture Pick isolated colonies
Media used: Falsely sensitive Inoculums too light
i. MHA – 3-5 mm deep; pH 7.2-7.4 results (larger ZOI) Very dry agar surface
ii. MHA with 5% sheep blood – for fastidious organisms (e.g. Agar not deep enough
S. pneumoniae) Falsely resistant Inoculums too heavy
iii. HTM – for H. Influenzae, MH supplemented with hematin, results (smaller ZOI) Too much moisture on agar
NAD and yeast extract Too deep agar
d. Place antibiotics on inoculated plates. Wait 3-5 minutes after Improper storage of Indicator of improper storage: Penicillin
streaking (do not wait >15 minutes). Use sterile forceps or disk & Methicillin (first to degrade)
antibiotic disc dispenser Reading and clerical In sulphonamides if there are 2 rings,
Placing of antibiotics: error measure outer ring
i. 90 mm plate: 8 disks Slight growth in zone of trimetroprim and
ii. 150 mm plate: 12-14 disks sulphonamides – disregard
iii. Disk 15 mm from edge of petri dish Swarming of proteus into zone – ignore
iv. Disks 24 mm away from center of any disks +
Increased Ca and causes ↑ resistance of P. aeruginosa to
e. Incubate plates in an inverted position (avoid accumulation of Mg++ aminoglycosides
moisture on the agar surface)
i. 35°C for 16-18 hours in ambient air E. Epsilometer test (E-test)
ii. 5-7% CO2: Strep, Haemophilus, Neisseria 1. Antibiotic strip with varying concentration of antibiotic along its
iii. Up to 24 hours for N. gonorrhoeae, MRSA, VRE length
f. Measure zone of inhibition 2. Elliptical zone of inhibition
i. Lawn of growth must be confluent or almost confluent 3. MIC: read where growth inhibition edge intersects
ii. Zones measured from back of plate F. Anaerobes Susceptibility Testing
iii. use caliper or ruler 1. Agar dilution method
iv. ignore faint growth of tiny colonies at zone edge a. Reference method (CLSI)
g. Interpret results b. Use supplemented Brucella laked sheep BA
i. Susceptible: infection can be appropriately treated with 2. Broth microdilution method
recommended dosage a. inoculum is 106 CFU/mL
ii. Moderately Susceptible: amount of antimicrobial agent can b. panels incubated anaerobically at 35° C for 48 hours
be increased beyond standard dosage 3. Etest
iii. Intermediate: susceptibility cannot be determined G. Automated Antimicrobial Susceptibility Test Methods
iv. Resistant: infection unlikely or will not respond to the 1. Optical approaches for examining results:
antimicrobial agent a. Turbidimetry: uses a photometer; turbidity = bacterial growth
b. Fluorometry: uses a fluorometer; degradation of fluorogenic
substrate = bacterial growth

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 16

 2. Automated Instrument Systems 4. Should be checked daily for presence of gas bubbles
a. VITEK 1, VITEK 2, and VITEK 2 Compact 5. Easier to read if permanently immersed in glycerol
b. BD Phoenix System C. Centrifuge
c. MicroScan WalkAway SI 1. Revolutions per minute (rpm) must be checked twice a year using
d. TREK Sensititre a tachometer
H. Special Antimicrobial Susceptibility Tests D. CO2 Incubator
Test Purpose 1. Percentage of carbon dioxide must be checked daily
Antimicrobial Measure the amount of antimicrobial agent E. Reagents
concentration test in serum or body fluid 1. Tested daily with both positive and negative controls
(assay) 2. Reagents that require QC are stains, antibiotics, biochemical
Minimum bactericidal Measure of the lowest concentration of testing, germ tube, typing sera, etc.
concentration (MBC) antimicrobial agent that kills a bacterial F. Personnel Competence
test isolate 1. All tests performed on patients must be subjected to proficiency
Serum bactericidal Measure of the highest dilution or titer of a testing twice a year
test (SBT) patient’s serum that is inhibitory to the 2. Participation in continuing education
patient’s own infecting bacterium and the G. Antimicrobial Susceptibility
highest dilution or titer that is bactericidal 1. Recommended control organisms are from ATCC
Synergy test Measure of the susceptibility of a bacterial 2. Daily tests with ATCC reference strain for 20-30 consecutive days.
isolate to a combination of two or more 3. If results are acceptable, frequency can be decreased to weekly.
antimicrobial agents 4. All results from the 20- to 30-day evaluation should be kept while
Time-kill assay Measure of the rate of killing of bacteria by the antimicrobial agent is being used
an antimicrobial agent as determined by 5. Antimicrobial solutions must not be refrozen after thawing
examining the number of viable bacteria 6. Strains used in QC of Routine Antimicrobial Susceptibility Tests
remaining at various intervals after Strains Purpose
exposure to the agent Staphylococcus aureus ATCC Drugs for Gram-positive
25923 bacteria
Escherichia coli ATCC 25922 Drugs for Gram-negative
XI. QA/QC IN MICROBIOLOGY and Pseudomonas aeruginosa bacteria
A. General Information ATCC 27853
1. QC is designed to ensure the clinical reliability of the laboratory data Bacteriodes fragilis ATCC 25285 Drug for anaerobes
2. QA includes maneuvers encountered in the three phases of Enterococcus faecalis ATCC Synergy screen tests
laboratory testing. 51299 positive
B. Thermometer Enterococcus faecalis ATCC Synergy screen tests
1. Checked periodically against a reference thermometer from the 29212 negative
National Institute of Standards and Technology (NIST) Haemophilus infuenzae ATCC H. Influenzae (non-B-
2. Temp used for calibration are -20ºC, 2-8ºC, 37ºC, and 56ºC 49247 lactamase)
3. Should be disposed if it differs by >1ºC

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 17

 Klebsiella pneumoniae ATCC ESBL (screening and b. Include manufacturer's verification in QC log.
700603 confirmatory tests) 2. For culture media prepared in the lab:
Neisseria gonorrhoeae ATCC N. gonorrhoeae B- a. 5% of each batch (maximum of 10) should be checked for
49226 lactamase negative sterility. Incubate for 48 hours.
Staphylococcus aureus ATCC B-lactamase positive for b. Check daily for moisture, sterility, color, hemolysis.
29213 MIC test c. Check with organisms expected to grow or give positive
Streptococcus pneumoniae Drugs for Streptococci reactions, and organisms not expected to grow or give negative
ATCC 49619 reactions.
7. Proper Storage of Antibiotic discs
a. Long-term storage: -20°C or below in a non-frost-free freezer
b. Working supply of disks: 2-8 °C for at least 1 week in a tightly FURTHER READING
sealed container with desiccant • Forbes, B., Sahm, D., and Weissfield, A. Bailey & Scott’s Diagnostic
c. Container should be warmed to room temperature before it is Microbiology 12th ed. USA: Mosby (2007)
opened • Brooks, G. Jawetz, Melnick & Adelberg’s Medical Microbiology 24th ed.
8. Antibiogram Boston: Mc Graw Hill (2007)
a. Overall antimicrobial susceptibility profile of a bacterial isolate. • McPherson, R. A., Msc, M. D., & Pincus, M. R. (2021). Henry's clinical
b. Used to verify the identification and susceptibility results. diagnosis and management by laboratory methods E-book. Elsevier
9. Cumulative Antibiograms Health Sciences.
a. Analysis of individual susceptibility results on isolates from a
particular institution in a defined period
b. % of isolates of a specie that is susceptible to the antimicrobial
(e.g., % of E. coli susceptible to ampicillin)
c. Purpose is to guide physicians in empiric therapy decisions and
for infection-control
H. Stock Cultures
1. Should be grown in large volume of broth and divided into vials to
last for a year
2. Stored in a freezer kept at -70C
3. Media used are the Schaedler broth with glycerol, chopped meat
(anaerobes), skim milk, tryptic soy agar deeps, and cystine tryptic
agar
4. To maintain viability of an organism, it should be subcultured twice
after thawing
G. QC for Culture Media
1. For commercially prepared culture media:
a. Exempt from testing if purchased from manufacturer who
follows NCCLS guidelines.

Microbiology | MODULE 3 | LABORATORY TESTING OF BACTERIAL INFECTIONS 18