Methods Of Studying

Bacteria
SEROLOGIC TESTING
1. Capsular reaction test or Quellung reaction
PURPOSE & SIGNIFICANCE
HISTORY
• first described by Neufeld in • gold standard method for
1902 pneumococcal typing
• utilizes a microscope and
specific pneumococcal antisera • to identify difficult isolates of
(ANTIBODY) presumptive pneumococci
Principle
• As a rapid method to definitively identify Streptococcus pneumoniae
directly in specimen, capsular swelling of the pneumococcus in
the presence of specific capsular antibody is observed.
(Bacterial capsule- antigen ; antisera- antibody against capsule)

• capsular swelling or halo

• India Ink counter stain (methylene blue)
• microscope The light transmitted through the capsule appears brighter
than either the pneumococcal cell or the background.
RESULT INTERPRETATION

Positive: Swelling of the capsules in

comparison to the control, giving a
sharply demarcated halo. Capsules
may be visible in the control but do
not produce a clear demarcation and
glassy appearance.

Negative: No appearance of a clear,

enlarged halo surrounding the stained
cell. No difference between the test
and control cells.
2. SLIDE AGGLUTINATION TEST
WIDAL TEST
Salmonella spp.
LATEX AGGLUTINATION TEST
The latex agglutination test uses latex
particles which are coated with anti-
cryptococcal antibody.
A visible reaction occurs when these
particles come in contact with a
patient sample that contains
cryptococcal antigen. LA can be used
on serum or CSF, and takes 20 to 45
minutes to perform. The major
benefit of LA is that it is over 90%
sensitive.
However, it can be expensive and
requires extensive laboratory
infrastructure, including refrigeration.
Antistreptolysin O
3. COUNTER-CURRENT IMMUNOELECTROPHORESIS
4. ELISA
An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and
measures antibodies in your blood. This test can be used to determine if you have
antibodies related to certain infectious conditions. Antibodies are proteins that your body
produces in response to harmful substances called antigens.
5. FLUORESCENT-ANTIBODY TESTS
• Bacterial antibody detected
through antibody labeled
with fluorescent dye
• Fluorescent chemicals are
attached to the constant
region of an antibody. If the
antigen is present, the
antibody binds to generate a
very specific, very sensitive
protein tag.
• Fluorescence microscopy
8. Microhemagglutination test
• based on hemagglutination of
erythrocytes sensitized with T.
pallidum antigen by antibodies
found in the patient’s serum or
plasma.

• It is used for both qualitative and

semi-quantative detection of
Anti-treponemal antibodies.
 9. Cold Agglutinin
• Antibodies agglutinate RBC at 4 dC
• Cold agglutinins are autoantibodies
produced by a person's immune
system that mistakenly target red
blood cells (RBCs).
• They cause RBCs to clump together
when a person is exposed to cold
temperatures and increase the
likelihood that the affected RBCs
will be destroyed by the body. This
test detects and measures the
amount of cold agglutinins in the
blood.
• Mycoplasma pneumoniae
MICROSCOPY
 BACTERIAL CULTURE
Principle: The process of growing bacteria in
vitro or in-vivo by providing the optimum
nutrients into its culture media.
universal precautions
1. barrier protection
2. engineering controls
-protect workers from hazards while performing procedures
-Biological Safety Cabnets (BSCs)- protect workers from aerosol
infection by sterilizing through several mechanisms
1. heat
2. ultraviolet light
3. High Efficiency Particulate Air (HEPA)- removes particles larger than
0.3 um
Biological Safety Cabinets (BSCs)
CLASS DESCRIPTION
CLASS I • Open- fronted, negative pressure, ventilated
• unsterilized room air enters and circulates within the cabinet
• exhaust air from the cabinet is filtered by a HEPA filter
CLASS II • also known as Laminar Flow Biological Safety Cabinet
• may have fixed opening (CLASS II A) or a variable sash opening (CLASS II B)
• Sterilize both air entering and circulating within the cabinet and exhaust air.
• routinely used by most hospital laboratory
CLASS III • entirely closed and all infectious materials are handled with rubber gloves that are
sealed in the cabinet
• all air entering and leaving the cabinet is sterilized with a HEPA filter
• supply air is drawn through HEPA filter while exhaust air is filltered through 2 HEPA
filters
• provides the highest level of safety
 TYPES OF BIOSAFETY LEVELS (BSLs) FOR INFECTIOS AGENTS
TYPE USUAL AGENTS PRECAUTIONS
1 • No known pathogenic potential for Adherence to standard laboratory
immunocompetent individuals techniques
• examples: Bacillus subtilis
• Most undergraduate laboratories operate under BSL 1
2 • imclude most common microorganisms associated • Level 1 practices plus laboratory coats,
with laboratory-acquired infection e.g. HBV, HIV, protective gloves, limited access,
Staphylococus, and Enteric pathogens (Salmonella decontamination of all wastes and
and Shigella) biohazard warning signs
• partial containment equioments Class I
and II BSCs
3 • air movement must be carefully controlled • Level 2 plus special laboratory clothing
• for handling Mycobacterium tuberculosis, Brucella, and controlled access
Coccioides immits, Rickersiae, Arboviruses
4 • primarily used in research facilities • level 3 plus entrance through separate
• exotic viruses- Filovirus and Arenavirus room where street clothing is changed
with laboratory clothing
• maximum containment
• decontamination of all personnel and
materials before leaving the area.
Culture Media 1. Liquid media
: Physical State ➢commonly called broths, milk, or
infusions
➢these are water-based solutions
that do not solididfy at
temperatures above the freezing
point.
➢these contain specific amounts of
nutrients but do not contain gelling
agenst such as gelatin or agar.
➢suited for the propagation of a large
number of organisms, fermentation
studies, and other tests.
Culture Media 2. Semi-Solid media
Physical State ➢ exhibit a clot-like consistency at
ordinary room temperature ans
contain agar at concetration of
0.5% or less that allows
thickening of the media without
producing a firm substance.
➢ they have a soft consitency
similar to custard and are best
suited for cultue of
microaerophilic bactera of for the
study of bacterial motility.
Culture Media 3. Solid media
Physical State ➢Agar – polysaccharide extract from
seaweed/algae
➢contain a solidifying agent such as 1.5%-
2% agar, givign them a firm surface oon
which cells can form discrete colonies
➢they are used for isolation of bacteria
and fungi of for determining the colony
characteristics of the organism under
study.
➢come in two forms:
a. liquefiable (or reversible) solid media
b. non-liquefiable (or non-reversible) solid
media
Culture Media 1. Synthetic media
Chemical State ➢contain chemically-defined
substances which are oure organic
and or inroganic compounds
➢the precis chemical composition of
a syntehtic media is known.
➢they may be simple or ocmplex,
depending on what supplement is
added to it.
Culture Media 2. Non-synthetic media
Chemical State ➢complex media that contain at leasr
one ingredient that is not chemically
defined, which means that it is
neither simple or pure compound.
➢it is not representable by an excat
chemical formula.
➢most are extracts of animals, plants,
yeasts
➢non-synthetic media can support
the growth of mor fastidious
organisms
Culture Media 3. Tissue culture – living tissues/
Chemical State immortal cells(cancer cells)
i. chick embryo/eggs
ii. A549 – from lung cancer
iii. Hela cell – cervical cancer
iv. Hep 2 cells – laryngeal cancer
 Culture Media: Functional Type
a. general isolation/supportive media-support the growth of
fastidious organisms e.g. nutrient agar and broth, trypticase soy
broth
➢ designed for primary isolation of a broad spectrum of microbes and contain a mixture of
nutrients that support the growth of both pathogenic and non-apthogenic organisms.
➢ ex. peptone water, nutrient broth, and nutrient agar
 Culture Media: Functional Type
b. enrichment media: encourage the growth of particular organisms, containing only a few of the desired
organisms amount large numbers of indigeneous flora e.g. Selenite F for S. typhi
➢ contain complex organic substances such as blood, serum, or psecial growth factors, and are designed to
increase the number of desired organisms without stimulating the rest of the bacterial population.
➢ used to grwo fastidious pr nutritionally exacting bacteria.
1. Blood agar - contains general nutrients with 50%-10% (by volume) blood added to a blood agar
base. Certain gram-positive bacteia produce exotoxins that cause hemolysis of red blood cells
containe in the blood agar. Their hemolytic reaction is categorized into three, which is useful in the
classification of these bacteria.
The hemolytic patterns are:
i. Beta hemolysis - shows omplete lysis of red blood cells resulting in complete clearing around
colonies.
ii. Alpha hemolysis - shows incomplete lysis of red blood cells, producing a greenish discoloration of the
blood agar aorund the colonies.
iii. Gamma hemolysis- shows no hemolysis, resulting in ni change in the medium.

1. Chocolate agar - a type of nutrient medium that is used for the culture of fastidious organisms such
as Haemophilus spp. Heat is applied to lyse the red blood cells, causing the medium to turn brown.
 Culture Media: Functional Type
c. Selective media- contains one or more agents inhibitory to all
organisms except the organism being sought e.g. Phenylethyl
alcohol agar (PEA), mannitol salt agar (MSA), Hektoen enteric agar
(HEA), Salmonella-Shigella agar (SSA)
1. Thayer-Martin- contains antibiotics trimethoprim, nystatin, vancomycin, and colistin. It is
used for the isolation of Neisseria
2. Mannitol Salt agar- contains 10% NaCl and used for the isolation of Staphylococcus aureus
3. MacConkey’s agar- promotes the growth of gram-negative bacteria, primarily those
belonging to the family Enterobacteriaceae, and inhibits the growth of grma-positive
bacteria through the addition of bile salts. Iti is both selective and differential.
4. Lowenstein-Jensen medium - a selective medium used to recover Mycobacterium
tuberculosis. It is made selective by the incorporation of malachite green.
5. Saboraud’s dextrose agar - used for the isolation of fungi
 Culture Media: Functional Type
d. Differential media- employs factors that allow colonies of
organisms that possess certain metbaolic or cultural charateristics
to be morphologically distinguished from other organisms e.g.
Sheep Blood Agar, MacConkey agar, EMB
➢ allow the growth of several tyoes of microorganisms.
➢ designed to show ivsible difference among certain groups of micrroganisms.
➢ differences may be in the form of cariations in colony size or color, changes in color of culture
media, or formation of precipitates or gas bubbles
➢ allow th growth of more than one target micrrosganisms that demonstrate morphologic
variations in colony morphology
➢ Ex. Mac Conkey’s agar and Triple Sugar Iron agar
 Culture Media: Functional Type
a. Transport medium- used if delay is anticipated.
➢ used specifically for organisms that need ot be transported to the laboratorately after collection.
➢ prevent the drying of specimen and inhibit the ovrgrwoth of commensals and contaminating organisms.
➢ Charcoal is added to neutralize inhibitory factors
➢ Ex. Cary Blair trnasport medium for transport of feces of suspected chilera patients and Pike’s medium which is used to
transport throat specimens of patients with streptococcal infection.
❖ Stuart's- increase vability of the organism, consists of a swab and transport medium activated by crushing an
ampule
❖ amies- modified stuarts medium
❖ sodium thioglycolate- can maintain bacteria up to 72 hours
❖ caryy- Blair- transport of stool specimen, for transport of enteric pathogens.
 Culture Media: Functional Type
a. Anaerobic media -
a. used specifically for organisms that cannot survive in the presence of oxygen and
require the reduce oxidation-reduction potential and other nutrients
b. supplemented with nutrients such as vitamin K and hemin
c. they undrgo boiling to remove dissolved oxygen
d. to reduce the oxidation-reduction potential, substance such as 1% glucose, 0.1%
thioglycolate, or 0.05% cysteiene are added.
e. methylene blue or resazurin is added as an indicator of the oxidation-reduction
potential
f. ex are chopped coat and thioglycolate
3. Dispensing/Distribution
1. Plated – dispensed on sterile petri
dish(EMB, Mac, BAP, CAP)
Prep: Weigh – dissolve – autoclave -
dispense

2. Tube – on sterile tubes

Prep: Weigh – dissolve – dispense -
autoclave
a. broth
b. agar slant
c. slant and butt
d. butt
Routine Culture Media
1. BAP – fastidious/hemolysis/defibrinated blood(V factor
2. CAP – Haemophilus/Neisseria/Isovitalex(X and V factors)
3. Thayer-Martin Agar(TMA) – Modified choc/antibiotics added
- N. gonorrhoeae/N. meningitidis
a. Vancomycin – G+
b. Colistin – G-
c. Nystatin– fungi(yeast)
4. Modified TMA – added trimethoprim(proteus inhibitor)
5. Martin Lewis agar – Neisseria/ anisomycin in place of
colistin
6. Eosin Methylene blue – lactose fermentation
- E and M indicators/ M inhibits G+
7. Mac Conkey – same use w/ EMB
- Bile salts and CV inhibits G+/ Neutral red(indicator)
8. Mannitol Salt agar(MSA) –G+ isolation/ CPS not CNS
- 7.5% NaCl/Phenol red indicator
9. Phenyl Alcohol agar – G+ isolation/PE inhib G-
10. Selenite Broth - S and S/ Na hydrogen selenite inhib most enterics
11. Xylose lysine desoxycholate - S and S/high NaCl inhib enterics
12. Thioglycolate – aerobes and anaerobes
Anaerobic Culture System
a. Resazurin –white(reduced) to pink(+)/used in PRAS
b. Methyene blue – blue to white/pink(+)
c. Catalyst – Palladium
Methods
1. Anaerobic jar - 5-10% CO2
2. Prereduced anaerobically sterilized system(PRAS)
3. Anaerobic glove box
Candle Jar Gas Pack jar
Method Environment

Aerobic culture O2 – 21%

CO2 – 0.03%

Candle jar O2 – 15%

CO2 – 5-10%

Anaerobic jar O2 – 0%
CO2 – 5-10%
H2 – 5-10%
N2 – 80-90%
Inoculation Techniques
1. Streaking
2. Semi quantitative – “dilution streaks: yields isolates from pure colony
Key characteristics – presumptive Dx, reduces cost, QC on automation
a. colony size(staph,strep,fungi),
b. Pigments - Staph, Pseudomonas,E. coli, Serratia, Salmonella(H2S+),
TSI(yellow)
c. shape/surface appearance(proteus,Klebsiella)
d. Hemolysis
e. Odor – pseudo(grapelike)
CULTURE:
1. SPECIMEN COLLECTION
2. SPECIMEN PROCESSING (different methods used in different
specimens)
3. Inoculation (Plating/Streaking)
4. Incubation of plates (24 hours @ 37℃) and (*BacTalert for Blood
Culture-)
5. Gram Stain bacterial colony
6. Biochemical methods (depends on G/S result)
Blood Culture
1. specimen collection
• venipuncture
• bactalert
• label bactalert with patient's
ID, date and time of
incubation

2. specimen processing (no

special processing)
3. Inoculation (Plating/Streaking)
• agar/media for blood culture:
• BLOOD AGAR PLATE (BAP)
• EMB or MacConkey plate
• Chocolate Agar plate (CAP)
• streaking/inoculation technique
(4 quadrants)
• shake bactalert first and draw a
small amount of blood using a
syringe
• place 1 drop of blood onto each
plate
• do streaking technique
• use different loops in every
quadrant
• purpose for this is to successfully
produce a well isolated colony
4. Incubation (24 hours @
37℃) (*Blood Culture-
BacTalert and plates)
• label the plates with patient's
ID, date and time of plating
• place all 3 plates inoculated
with the specimen/ blood
sample inside an incubator
for 24 hours @ 37℃
• place bactalert inside the
incubator 24 hours @ 37℃
 after 24 hours Blood agar plate
• remove agar plates from the incubator & examine
agar plate in the biosafety cabinet
• check the plates for growth & Record all your
Notice the 4
observations: quadrant pattern
• hemolysis (a, b, g)
• quantity of growth of bacteria
• few, moderate, heavy
• color of growth
• colorless, creamy, white, pink, black, green, red, etc
• consistency of growth
• dry, shiny
• colony size
• pinpoint, small, medium, large
• shape
• round, irregular, swarming Blood agar plate
• other special characteristics
Notice the well
ex. heavy growth of large, round, shiny,
creamy, gamma hemolytic (*non-hemolytic) isolated bacterial
colonies of ________________ colonies on the 4th
after 24 hour-incubation period @ 37℃ on quadrant
Blood agar plate.
 after 24 hours in EMB agar
Notice the unique
• Record all your observations like color seen in the
what we did in BAP grwoth of bacteria

• EX. heavy growth of large,

round, shiny, green, & green-
metallic sheen colonies
__________ after 24 hour-
incubation period @ 37℃ on
EMB agar plate.
after 24 hours in Mac Conkey agar
• Record all your observations like
what we did in BAP
• heavy growth of large, round,
shiny, creamy-light pink colonies
of _____________ after 24
hour-incubation period @ 37℃
on Mac Conkey agar plate.
Staining: Differntial Stains
• stains are used to differentiate one group of bacteria from another.
• There are two tyopes of differential staining procedures commonly
used, namely:
• Gram Stain
• Acid-Fast Stain
5. Staining: Differntial Stains: G/S
• distinguishes gram-positive bacteria
from gram-negative bacteria
• Gram positive bacteria stain blue or
purple, while gram-negative bacteria
stain red or pinl
• As a general rule, all cocci are gram-
positive except Nesseria, Veillonella,
and Branhamella
• On the other hand, all bacilli are
gram-negative except
Corynebacterium, Clostridium,
Bacillus, and Mycobacterium.
5. Gram stain or G/S
• choose a well isolated colony for
bacterial smear
• heat fix the bacterial smear by
passing it onto a flame three
times.
5. Gram staiStaining: Differntial Stains
n or G/S
• Table
• Reagensts used in Gram-staining
and expected results
• G/s procedure
5. Gram stain or G/S
• results:
• G+C
• G-C
• G+B
• G-B

• +4 gram
negative bacilli
5. Staining: Differntial Stains: Acid-fast stain
• stain used for bacteria with high
lipid contet in their cell wall,
hence cannot be std using Gram
stain.
• two methods are used, namely:
• Ziehl-Neelsen stain
• Kinyoun stain
5. Staining: Differntial Stains: Acid-fast stain
• Ziehl-Neelsen stain • Kinyoun stain
• also known as the “hot method” • also known as the “cold method”
• it requires steam-bathing the • does not utilize heat after the
prepared smear after the addition addition of the priimary stain, which
to the primary dye. is oil-based
• this is because the primary stain • acid-fast organisms will appear red
used is aqueous and will not bind on a green background
to the cell wall of the organism
• acid-fast organisms will appear red
on a blue background
5. Staining: Differntial Stains: Acid-fast stain
• table • table
• Reagent sused in acid-fast staining
and the expected outcomes
• Procedures in proper acid-fast
staining
5. Staining: Special Stains
• these are used to demonstrate • pictures
specific structures in a bacterial cell
• for instance, methachromatic granules
can be visulaized using the LAMB
(Loeffler Alkaline Methylene Blue) stain
• Other special stains:
• Hiss stain (capsule or slime layer); Dyer
stain (cell wall), Fischr-Conn stain (flagella),
Dorner and Schaefer-Fulton stain (spores),
and India inl or nigrosine (capsule of the
fungus Cryptococcus neoformans).
6. Biochemical methods (ex. G- Bacilli in all 3
agars)
1. Oxidase test (enzyme)
(ex. Oxidase +)
BAP (choose BA for Oxidase) EMB MacConkey
2. biochemical testing (sugar
fermentation and others)

1. Triple Sugar Iron (TSI) or

Kligler Iron Agar (KIA)
2. sulfide-indole-motility (SIM)
3. Citrate
4. Urease
5. Lysine Iron Agar (LIA)
6. Methyl red-Vogues Proskauer
(MR-VP)
Over-all results: E. coli
1. G/S: G+Bacilli 1. 4. Triple Sugar Iron (TSI) or Kligler
Iron Agar (KIA) : yellow/yellow;
acid/acid- lactose fermenter with
2. Colony growth: gas
BA: non-hemolytic 2. sulfide-indole-motility (SIM): -/+/+
3. Citrate: - (neg)
EMB: green-metallic sheen
4. Urease: - (neg)
MaC: pink-lactose fermenter
5. Lysine Iron Agar (LIA): +/+ with
FeCl3
3. Oxidase + 6. Methyl red-Vogues Proskauer (MR-
VP): +/-
API system or analytical profile index: classification of
bacteria based on biochemical tests, allowing fast
identification.
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Learning Objectives
At the end of the lesson, the student should be able to:
1. discuss the importance of antimicrobial susceptibility testing;
2. interpret results of antimicrobial susceptibility testing;
3. classify antibiotics based on mechanism of action;
4. describe the characteristics of an ideal antibiotic; and
5. discuss the mechanisms of drug resistance.
Antibiotics are given to treat infectious diseases. The physician faces
the problem of decidding which antibiotic to use for a given infectious
diseases. To make sure that the antibiotic to be given is suited for a
specific organism, an antimicrobial susceptibility test must be
requested. This test will tell the physician if the organism involved in
the disease process is susceptible or resistant to a particular antibiotic,
thereby saving the patient from spending money on a drug that not
work on the particular organism involved in the first place.
Susceptibility testing is most often indicated when the etiologic agent
involved is known to be capable of developing resistance to commonly
used antimicrobial agents. It is rarely done if the organism is not known
to develop resistance against a given antibotic.
There are two methods used in susceptibility testing- the disc diffusion method adn
the test tube method. The test tube method is a serial dilution that is tedious and
time-consuming. It is used to determine the bacteriostatic and bactericidal
concentration of antibiotic. The method routinely used in most laboratories is the
disc diffusion method. In this method, paper discs are impregnated with known
concetration o different antibiotics. These are then plaed on agar plates where the
microorganism has been inoculated. It is relatively simple to do and interpret.
Interpretation is done by measuring the zone of inhibition around the colonies.
However, there may be variations in results which may be due to several factors,
namely:
1. the size of inoculum
2. the of antibiotic molecule
3. length of incubation
The disc method can be used to determine bacteriostasis only. It is capable to fast-
growing aerobes and facultative microorganisms.
Materials
• Prepared cultures of Staphylococcus aurues and E. coli
• Commercially prepared antibiotic discs (Penicillin, Chloramphenicol,
Tetracycline, Methicillin, Erythromycin). Other antibiotic discs may be
used if available.
Procedure
1. On the culture plates, place equidistant from each other and in a
circular fashion, one disc each of the commercially prepared
antibiotic discs.
2. Incubate at 45 degree Celcius for 16-18 hours.
3. After the incubation, measure with a ruler the widest diameter of
zomes of inhibition of each antibiotic (expressed in millimeters).
4. Compare the measurements obtained with the reference table.
Record your result with the interpretation (susceptible,
intermediate susceptibility, resistant).
 ACRONYMS
• MRSA- METHICILLIN-RESISTANT S. aureus
• VISA- VANCOMYCIN (GLYCOPEPTIDE)-
INTERMEDIATE S. aureus
• VRSA- VANCOMYCIN-RESISTANT S. aureus
• VRE- VANCOMYCIN R Enterococcus faecium
• ESBLs - Extended-spectrum b-lactamases
• GRAM-NEGATIVE RODS
Agar Diffusion Tests
a. Disk Diffusion test (Kirby Bauer)
-most widely performed
-Principle: Disks with variety of antimicrobials are applied to the surface
of Mueller-Hinton Agar that have been inoculated with organism.
Incubated at 37 dc for 24 hours, then zone of inhibition or ZOI is
measured.
-done on a pure colony of the identified bacteria
Materials
O.5 McFarland standard
Mueller-Hinton agar
Forceps
Caliper or ruler
Sterile swab
Inoculating loop
Antibiotic disks
Bunsen burner
Incubator (37 ℃)
Vortex
Pure colony of identified bacteria (24 hour-
incubation)
procedure
 SIR INTERPRETATIONS
• SUSCEPTIBLE (S) (mm)
• INFECTION BY THE STRAIN MAY BE APPROPRIATELY
TREATED WITH THE DOSE OF ANTIMICROBIC
• INTERMEDIATE (I) (mm)
• RESPONSE RATES MAY BE LOWER THAN FOR SUSCEPTIBLE
ISOLATES
• RESISTANT (R) (mm)
STRAINS NOT INHIBITED BY THE USUALLY ACHIEVABLE
SERUM CONCEN OF THE AGENT WITH NORMAL DOSING
 ANTIMICROBIAL DRUGS
• Broad Spectrum Antibiotic:
• Has actions against both gram positive and gram negative
bacteria, ex. Tetracycline
• disadvantage: Inihibition or destruction of normal flora

• Narrow spectrum antibiotic

• has a limited spectrum of action, ex. Penicillin is only
effective against gram positive organisms
 MECHANISM OF ACTION SITE OF ACTION AGENT
1. Inhibition of cell inihibits cross-linking of Penicillin, Cephalosporins,
peptidoglycan layer Carbaenems, Aztreonam
wall synthesis
inhibits other steps in cycloserine, vancocin, bacitracin
peptidoglycan synthesis
2. Inhibition of 30 s ribosomal unit Tetracycline, Gentamicin,
Aminoglycosides
Protein Synthesis
50 s ribosomal unit chloramphenicol, erythromycin.
clindamycin

microtubule function griseofulvin

3. inhibition of nucleic inhibition of nucleotide sulfonamides, trimethoprim

synthesis
acid synthesis quinolones,
inhibition of DNA synthesis
rifampin
inhibition of mRNA synthesis
 MECHANISM OF SITE OF ACTION AGENT
ACTION
4. alteration of cell bacterial membrane polymyxins
membran function permeability
amphotericin B,
fungal membrane Ketoconazole
permeability
5. inhibition of mycobacterial mycolic isoniazid
mycolic acid synthesis acid synthesis

6. uncertain uncertain site of metronidazole,

mechanism action pentamidine
Answer the following:
1. What are the characteristics of an ideal antimicrobial agent?
2. List down the different antibiotics used or the exercise and give the
mechanism of action.
3. What is meant by resistance? Give the mechanisms by which
organism develop resistance.
END OF LESSON