Done by: Sumiya Talal

Al harbi

Under supervision of
specialist: Majed
Abdulhamed Alshaer

KING ABDULLAH
MEDICAL CITY

HOLY MAKKAH

30, NOV2016
WELCOM TO
MICROBIOLOGY FOR MORE
Internship year INFORMATION
This book provides a basic information about microbiology section CONTACT:
which collected from different resources. Somayah-
1415@hotmail.com

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Table of Contents

Introduction about microbiology ................................................................. 4

Methods of identification: .....................................................................................................5
(basic bacterial classification) ..................................................................... 17
Microbiology section in KAMC: ................................................................... 19
First: reception and receiving area ..................................................................................... 22
Second: respiratory bench .................................................................................................. 24
Third: blood bench area ...................................................................................................... 28
Forth: urine and stool bench .............................................................................................. 29
Classification and testing of Antibiotics ...................................................... 33
Antimicrobial susceptibility testing (AST) ...................................................35
Minimum inhibitory concentration (MIC) .................................................. 36

E Test Method: ............................................................................... 37

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Objectives:

-To know the basic of bacteriology including, taxonomy, bacterial classification,

different diseases, morphological and microscopic identification of bacteria.
-To understand the type of pathological and normal flora of bacteria.
- To be able to perform all the practice in each bench of microbiology.
-To understand the mechanism of action of antibiotics against each bacterium.
-To know the principle of instruments used in microbiology section.

The message:

To facilitate the practical and theoretical information and handled to each intern.

Acknowledgment:

I would like to thank Mr. Majed Al shaer for helping the preparation of this booklet and
providing helpful suggestions and encouragement throughout our internship and our life in
general.

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 Introduction about microbiology

Microbiology is:

study of living organism which can't be seen by necked eye and

seen with the help of microscope.

Taxonomy is:

The since of biological classification including, nomenclature and

identification of organism.

Classification is:

the arrangement of organisms into groups (e.g. families, genera) on the

basis of similarities or relatedness in their various characteristics, which

includes:

— Morphology

— Staining properties

— Cultural characteristics

— Biochemical reactions

— Antigenic structures

— Genetic relatedness

Morphology of bacteria:

Cell Shape:

-Bacteria have a wide variety of sizes and shapes.

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- bacteria that possess cell walls exist in three basic morphologic forms:

 Spherical (round) cells or cocci

 Rod shaped cells or bacilli

 Spiral shaped cells or spirilla

Cell Size:

Bacteria are 0.2 to 2 μm in diameter and 1-6 μm in length.2

Methods of identification:

BIOCHEMI
CULTURE
MICRO CAL SEROLOGIC MOLECULA
CHARACTER
SCOPIC REACTION TESTS R METHODS
ISTICS
S

1- Microscopic identification:

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 Unstained Stained
Preparation Preparations
Staining
Wet Mount Methods

Gram’s Ziehl- Giemsa

Simple Neelsen Staining Albert’s Fluorescent
Staining
Staining
for e.g.coryneb
methylen mycobactiru acterium
e blue& m tuberclosis sp.
carbol
fuchsin Negative Spore Flagella
Staining

1-Unstained (wet film) procedure: usually used to identify the fungi from
bacteria.

40X power

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 2- Stain procedure (gram staining):
*The Gram’s stain divides the bacteria into two groups:

-Gram-positive bacteria (Blue or purple colour)

-Gram-negative bacteria (Pink or red colour)

*The Gram stain requires the use of:

1- Primary stain - Crystal violet

2- Mordant – Iodine

3-Decolourizer - Alcohol and

4- Counter stain - Safranin

Procedure:

1 mint 1 mint seconds 1 mint

*Function of iodine: fix the crystal violet on the wall of bacteria.

Factors affecting the result of gram staining:

-Over depolarization -improper fixing the smear

-over heating - the freshness of the reagents

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Gram staining result:

Positive gram negative gram

stain seen stain seen
under 100x oil under 100x oil

2- ziehl-neelsen staining:
Principle: Mycobacterium species, are coated with a thick lipid material
(e.g. mycolic acid) in their cell walls and keep the basic dye (basic fuchsin)
despite of acid alcohol treatment and considered as acid fast (AFB).

Procedure:

1 mint seconds 2 mint

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Result seen under 100x oil immersion:

Acid fast bacilli >> commonly sample used is sputum to see

mycobacterium tuberculosis that cause TB.

3- Culture media:
An artificial culture medium should contain all the nutrients i.e.

-sources of carbon

-sources of nitrogen

-sources of minerals

-sources of essential vitamins and water that are needed by the bacteria
for its growth and multiplication in the laboratory.

Forms of culture media:

-Solid media(agar)

-Liquid (broth)

-Semi-solid media

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Type of media indicator information pictures

SAB (Sabouraud _____ -Used for fungal culture

Dextrose Agar) -contains antibiotics that
inhibits growth of other
organisms including:
chloramphenicol&
gentamicin
- low PH acidic media
Blood agar _____ -Type of enriched media
which specific substance is
added
- shows type of hemolysis
which are:
Alpha> partial hemolysis
Beta > complete hemolysis
Gama> no hemolysis

XLD agar _____ -Type of selective media

(Xylose lysine which select the growth of
desoxycholate) salmonella and shigella and
inhibit the growth of others.
- salmonella produce a black
colony due to production of
H2S
- shigella will be colorless on
agar
-sample used: stool

Todd Hewitt ____ Used as a culture media for

broth streptococcus agalactia in
maternal infection.

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Chocolate agar ____ Type of enriched media that
contain lysed RBCs, X and V
factors that required for H.
influenza growth.

Mannitol salt Phenol red -Type of selective media for

agar staph. Aureus.
- used for MRSA screening
test (methicillin resistant
staphylococcus aureus).
-nowadays, oxacillin used
instead of methicillin.
-MRSA produce a yellow
colony on mannitol agar due
to fermentation of mannitol.
- Other staph produce a
pink color.
-contain a high
concentration of salt 6.5%
which inhibit other bacterial
growth.
- if staph grow in culture this
indicate resistant to
oxacillin.
- sample used: groin, axilla
and nasal swab.

MacConkey's Neutral red -Type of selective media for

agar gram negative bacilli
bacteria and differential
media for lactose fermented
(pink color) and non-lactose
fermented organism (yellow
color).
-contains: crystal violet and
bile salt which inhibit growth
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 of gram positive bacteria
- proteus can grow but it will
not produce swarming due to
bile salt.
- used for CRE (carbapenem
resistant enterobacteria),
CRP (carbapenem resistant
pseudomonas) and
Acinetobacter resistant.

Selenite broth ______ -Enhancement media for

salmonella and shigella
growth.
-after 24h of incubation a
sample taken from selenite
and culture on XLD agar this
called sub-culture process.

Bile esculin agar _____ -Type of selective media for

enterococcus organism
which produce a black color
on agar.
- used for VRE screening test
(vancomycin resistant
enterococcus)
-sample used: rectal swab.
-Mechanism of action: the
organism will react with
esculin hydrolysis
Product+ ferric iron
black color.
Thioglacte broth ______ Used for anaerobic organism ______
Sample used: body fluid.

Thiosulphate Bromothymol Type of selective media for

citrate bile salt blue vibrio's and differential
sucrose (TCBS) media which differentiates
agar sucrose fermenting (yellow
colonies) from non-sucrose
fermenting Vibrio's.

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 -V. cholera produce yellow
colony
- V. Para hemolytic produce
colorless colony.
-TCBS contain sodium
thiosulfate which inhibit
other bacterial growth.
- alkaline PH.
Molar Hinton _____ -Simple media used for
agar manual sensitivity antibiotic
test.
-ingredient: beef, peptone
and starch.
Used for H. influenza sp by
utilizing X, V and XV factors.
- X factor> hematein
- V factor >NAD
(Nicotinamide adenine
dinucleotide)
-H. influenza & H. aegyptius
utilize both V&X factors
-H. parainfluenza utilize only
V factor
-H. ducreyi utilize X factor

Thayer martin _____ -Selective media for

agar Neisseria sp.
-contains antibiotics that
inhibits other bacterial
growth including:
Colistin> to kill gram
negative bacteria
Vancomycin> to kill gram
positive bacteria
Nystatin> to kill fungi and
yeast
Sulfa methylene
and\ trimethoprim to inhibit
proteus growth.

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 Ingredient: molar Hinton,
chocolate and 5% of
antibiotics.

Einstein lactose Bromothymol -Used for urine culture

electrolyte defect blue -Selective for Enterobacter
(CLED) and E, coli which produce a
yellow shadow

Chrome agar -For colorful microbial

detection.
-Used mainly for multi drug
resistant organism
including:
Acinetobacter> produce
green color
VRE> red color
CRE> purple color

-Biochemical test

Test for enzymes including:

1- Catalase test 2- Coagulase test

3-Oxidase test 4-Urease test

Catalase test:

Principle:
To determine the production of catalase enzyme by the organism, which
breaks down H2O2 into H2O and O2.
Control Organisms:
+Ve control – Staphylococcus aureus
-Ve control – Streptococcus species

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Result: Rapid production of (bubbles).

Coagulase test:

Principle:

To test the ability of an organism to clot plasma by coagulase enzyme.

Two type of coagulase test:

Free coagulase (tube test): which converts fibrinogen to fibrin by activating coagulase-
reacting factor present in plasma.

• not commom used

Bound coagulase (slide test): which converts fibrinogen directly to fibrin without requiring
coagulase-reacting factor . It is detected by clumping of bacteria in the rapid slide test

• commonly used

Control Organisms:
+Ve control – Staphylococcus aureus
-Ve control – Staphylococcus epidermidis

Result: positive negative

Oxidase test:

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Principle:

To detect the presence of oxidase enzyme, which oxidizes cytochrome by

molecular oxygen.

Interpretation:
Positive: deep purple within 10 sec.
Negative: No color change.

This test used to identify gram negative non-lactose fermented bacteria

+VE >> pseudomonas

-VE>> Acinetobacter & protues

Result:

Urease test:
Principle:

To detect the ability of organism to produce the urease enzyme, which

splits urea to ammonia and carbon dioxide.

Interpretation:
Positive – Pink color >>Klebsiella sp, Proteus spp., Helicobacter pylori
Negative - Pale yellow>>Escherichia coli
negative positive
Result:

(basic bacterial classification)

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 bacteria classification based on gram stain reaction

Gram positive Gram negative

bacilli cocci bacilli

cocci

Neisseria Moraxella H. influenza

Enterbacteriac
streptococcus staphylococcus
eae
Corynebacterium
Catalase test
diphtheria (normal
flora) on skin

negative strep Positive staph

Coagulase
test

Positive Negative
Novobiocin test
>staph.aures (CNS)

Resistant>s. saprophytic Sensitive> s. epidermis

cause UTI infection (normal flora)

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 streptoccocus

classified
according to
hemolysis

alpha beta gamma

hemolysis hymolysis hemolysis

greenish color on classified Group D

blood agar due to according to
conversion of Hg to> lancefield group Enterococcus
methemoglobin to:

- hemolytic, Bile
optichin
sensetivity test aesculin hydrolysis
Group A> s. pyogenes-
Positive. Small gray
pinpoint on blood agar. -
colony on blood agar,
sensitive> s. wide zone of hemolysis. -
pneumonia
and black on bile
causes pharyngitis.
(mobile on bile aesculin agar.
solubility test) Bacitracin test> sensitive

resistant > s.
viridans( Group B> s. agalactia
normal flora) normal flora in female
genital tract. - big colony on
blood agar. -narrow zone of
Staph. aureus hemolysis. Bacitracin test>
resistant

(CAMP) Identification test for s. agalactia.

Test showing the arrow-shaped zone of
enhanced hemolysis (positive result)
of Streptococcus agalactia (group B)

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 The streak plate technique:

A streak plate technique is used to obtain single isolated colonies from a clinical
specimen (sample).

1
2
4
3

After incubation, single isolated A small portion of sample is taken

colonies should be visible within the on a sterile loop and streak on an
fourth zone. agar medium into four zones,
reframing the loop between zones

Microbiology section in KAMC:

Instruments used in microbiology section:

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 instrument information pictures

Bactec 9240 -Used for blood culture.

- aid in diagnosis of
bacteremia.
- blood bottles are used as a
container for blood
samples.

Blood culture bottles -There are two type pf

bottles:
AEROBIC (blue cover) and
anaerobic (purple cover)
-It contains:
1-SPS (sodium polyantha
sulfonate) that act as
anticoagulant, prevent
phagocytosis and
complement reaction
(liquid part)
2- a sensor (the gel on the
bottom) to detect bacterial
growth that secret co2
during multiplication it
gives alarm when growth
detected.
3-Resin or beads: acts as
neutralization and
absorption of antibiotic. -
The amount must be from
8-10ml for adult and 1-5 ml
for children.

Microscan walkaway 96 -Aid in identifications of

bacteria and play an
essential role for antibiotic
sensitivity test (AST).
-Principle: colorimetric it
detects the change in color
due to different biochemical
reaction related to specific
bacteria.

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Vitek 2 compact -Aid in identifications of
bacteria and play an
essential role for antibiotic
sensitivity test (AST).
-Principle: colorimetric it
detects the change in color
due to different biochemical
reaction related to specific
bacteria.
Component of vitek -an identification cart (kit)
used for bacterial
identification whether gram
positive or negative or fungi
- each kit used for specific
organisms.
-It contains all biochemical
reaction according to
isolated bacteria.
- other kit used for (AST).

Rack used for kits

General purpose -for fungal, bacterial growth

incubator. -each incubator has specific
temperature for growth
depending on the type of
organism.
Bacteria growth in>> 36c
while fungal in>> 25c
-The duration of bacterial
growth takes from 24h to
48h
-Fungal from 1 week to 4
weeks

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Class II biohazard Used for culture purpose to
safety cabinet. insure the safety for the
specialist and removing any
contamination from the air.

Auto-flow IR water Some bacteria need CO2 for

jacketed CO2 incubator. growth so this type of
incubator provides the
suitable amount of CO2 to
each bacterium.

Microbiology divided into five sections:

1-reception and receiving of clinical samples

2-respiratory bench

3-blood & body fluid& tissue bench

4-urine and stool bench

5-mycology bench

First: reception and receiving area

This area receives different type of clinical samples include:

1-Urine 2- stool 3- blood 4- tissues 5- sputum 6-BAL

7-rectal swab 8- throat and mouth swab 9- eye and ear swab

10- wound swab.

How do we receive samples?

According to acceptance and rejection criteria which is:

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 Rejection criteria:

1-All unlabeled samples must not receive.

2-all miss labeled samples must be verified before receiving.

3-old samples must be rejected.

4-unseffecient mount of samples must not receive and send a request to

gather another sample.

5-unsterile container.

6-if there is a leakage of samples inside the biohazard bag.

7- if the sample is sputum and there is a saliva more than the accepted we
must rejected because it contains an epithelial cell that interfere with the
result.

8- tissue samples inside a formaldehyde container because, the

formaldehyde will kill any present bacteria and we need it for the
diagnosis.

9- needle with an aspiration samples, recap of needle is not allowed for

safety purpose.

Which type of media used with the different samples:

CULTURE Direct examination

specimen BA BA MAC CHOC SAB BA\MAC Thioglcate Gram Wet KOH
aerobic anaerobic broth stain preparation

Ear     * * 
Nasal     *
secretion
Throat  
Mouth   *
sputum    *  *
BAL    *  *

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eye    *  *
Skin   *  *
tissue    *   *
Catheter  
tip
Wound     
swab
Pus    *  
aspirate
Body     *   *
fluid
CSF    
urine *3   *
HVS   * 
MRSA Blood and mannitol salt agar with oxacillin
VRE Blood and bile esculin agar
stool XLD and selenite broth

Key: (*) when fungal infection is suspected, (*3) when yeast cells seen in
urine examination.

Note:

Each day you must check the quality control of each machine including,
biohazard class II safety cabinet, microscopes, stains, general purpose
incubator, refrigerator temperature and media stability.

Second: respiratory bench

In this area, we must know the most common pathological bacteria
present in respiratory tract and these bacteria classified according, the
site of infection to:

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 Upper and lower tract infection:

Nose:
Mouth:
staphylococcus
spp yeast

Throat: Nasopharyngeal:
streptococcus. mortadella spp
pyrogen

Lower tract includes:

-H. influenza

-Moraxella

-s. pneumonia

Morphological characteristics of bacteria on agar:

-H. Influenza is growing on
chocolate agar. Notice semi-
opaque, gray-white, mucoid.
-Gram negative- small- cocci
-Oxidase + catalase+ Aerobic
-Moraxella -Gram negative- short
rods coccobacilli- cocci
-Colonies appear as Smooth with
a grayish Wight color.
-When the colony pushed with
loop they scoot a cross the media.
-strep. pneumonia -Gram
positive- cocci with alpha
hemolysis.
-Colonies appear as mucoid green
in color.
-bile solubility test +

-Oxidase + catalase+

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-strep. pyrogen Gram +ve cocci
arranged in chains
-Aerobic, non-motile and produce
grey-white colonies.
- Pin point colonyies with Wide
zone of hemolysis.
-staph. aureus Gram +ve cocci
arranged in clusters -yeast appears like star in shape
- coagulase positive Wight off in color.
-Aerobic, non-motile, produce
golden yellow colonies

- -haemolytic

Significant and insignificant growth of bacteria:

According, to the number of organism on the streak if:

1-Low number of organism grow in one streak considered as normal flora or

insignificant growth. > the result is normal

2-moderat number of growth in media there are three cases:

- If one type of organism grow considered a pathogen and we must use the Vitec
machine for identification.

-If two type of organisms grow on media (the many in number) > pathogen and (low)
> insignificant.

-If three type pf organisms grow considered as mixed and we must reject the sample>
no farther work out.

Screening test:
-MRSA screening considered an important test for inpatient to monitor their health.

-MRSA grow on mannitol salt agar as we mentioned before.

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Gastrointestinal (GTT) test:
-For yeast identification

-sample used (serum or plasma)

Procedure:

1-apply the wet mount technique by taken a small sample from the agar an place it on
microscope slid then cover it with a cover slip.

2-see it under 40X power

3-if the organism appears like a pudding shape perform the GTT test

Pudding
shape

4-take enough colony and add it into tube contains serum.

5-incubate the sample in general incubator for 2-3 hours.

6-see the result after perform the wet mount on 40X.

Result:

Candida Albicans
germ tube

-If other organism is different from the germ tube right (other than candida albicans)

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 Third: blood bench area
When the blood cultivation is needed?

In case of:

- High temperature of unknown cause

- Endocarditis which caused by inappropriate tooth extraction and the causative

organism will be s. pyridines

-No antibiotic administration

-The amount of sample (blood) that will be collected from adult must be from 8-10ml

-While children the mount arrange between 1-5 ml

-Blood are collected in special container called (blood culture bottles) as we

mentioned before.

-after bottles monitoring they placed in BACTEC machine for processing

and identification of the positive organism.

-The duration of result takes about 5 days if there is no alarm sound for positive
result we considered it as negative (normal sample).

Steps to deal with the positive results

If the positive result was in Aerobic If the positive result was in Anaerobic
bottles take it and do the culture process: bottles take it and do the culture process:

-blood agar -blood agar O2

-chocolate agar -blood agar CO2

MacConkey's agar -chocolate agar

-gram stain MacConkey's agar

Note: All the samples must be sterile -gram stain

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 Bacterial present in CSF samples according, to the age

Children: Newborn:
Adult and elderly:
H. influenza S. agalactia
-s. pneumonia
type B E. coli
- Neisseria
- nectria monocytogen

How can we differentiate between bacterial and viral infection?

By three ways:

1-gram staining technique.

2-cell count (bacterial infection indicate a high number of neutrophils,

viral infection indicates a high number of lymphocyte count, TB infection
both will be high).

3-glucose concentration (bacteria will consume it so the amount will be

low, in viral infection glue> normal.

Forth: urine and stool bench

In this area, we are looking for family of Enterobacteriaceae I & II.

General characteristic's:

1-Found in the GIT of humans and animals.

2-Gram negative, non-spore forming.

3- Aerobic and facultative anaerobes.

4-Grow in blood and MacConkey's agar.

5-Ferment glucose and produce acid.

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Enterobacteriaceae I (lactose fermenting):

-Escherichia
-Klebsiella
-Enterobacter
-Citrobacter

Enterobacteriaceae II (non-lactose fermenting):

-Salmonella -Morganella

-Shigella -Proteus

- Serratia

MacConkey's agar plate

Infection by Enterobactericeae:

Morphology of most common bacteria:

1-E. coli: is part of normal flora of intestine of humans and

animals.

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-Aerobic, Gram-negative bacilli.

-Produce pink colonies (by fermenting lactose) on MacConkey agar.

-Motile, non-spore formers.

(characterized by pink shadow)

2- Klebsiella:

-Aerobic, Gram-negative bacilli. E. coli on MacConkey

agar
-Produce large, mucoid colonies on blood

and MacConkey agar and ferments lactose.

-Non-motile, non-spore formers.

-Urease +ve after 18-24 hours.

klebsiella on klebsiella on blood

MacConkey agar agar

3-salmonellae: are motile, aerobic, gram-negative bacilli.

-Do not ferment lactose.

-Produce H2S from Sulphur-containing amino acids.

-Grows on XLD agar and produce black colony

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4- Shigella:

-non-motile, aerobic, gram-negative bacilli.

-Do not ferment lactose.

-Do not produce gas during carbohydrate fermentation.

- appear as pink to red colony on XLD agar.

5-proteus:

- Produce Swarming growth on blood agar.

-NLF colonies on MA, DCA and SS Agar. Swarming is inhibited on these

media due to presence of bile salts.

Some immportant NLF bacteria:

Pseudomonas:
Acinetobacter:
1-G –ve rods, strict aerobes, and motile.
Non-fermenter, Oxidase positive. 1-G –ve rods, nonmotile. Non-
fermenter, Oxidase negative.
2-Fruit smell and produce green pigment
on MacConkey and blood agar due to: 2-regular circle colony
Thiocyanide day and pyoverdine day
production

Acinetobacter on
Pseudomonas on MacConkey agar
Pseudomonas on blood
MacConkey agar agar
32 | P a g e
 Classification and testing of Antibiotics:

Antimicrobial agents can be classified according to their mode of activity:

-bacteriostatic: inhibit bacterial growth – bactericidal: kill bacteria

Classes of antibiotics:

Cell wall
synthesis Cell membrane
inhibitors protein synthesis
Nucleic acid inhibitors
synthesis
synthesis
inhibitors
inhibitors
Beta-lactams antibiotics
include: 1-polymyxins:
1-Aminoglycosides
1-cephalosporins include: 1-Sulphonamides -polymyxins E
include: include: (colistin)
-Streptomycin
-cefepime -Sulfamethoxazole -polymyxin B
-Amikacin
-cefotaxime -Trimethoprim
-Gentamicin
-ceftazidime 2- Quinolones
2- phenicol's include:
2- penicillins include: (Nalidixic)or
- Chloramphenic Fluoroquinolones
- Penicillin p ol include:
- Ampicillin
3-Tetracycliness - 1-Levofloxacin
- Oxacillin
include: - 2-Norfloxacin
- Pipracillin
- Tetracycline - 3-Ciprofloxacin
Other beta lactams
- Doxycycline 3-Rifamicins include:
(imipenem&
meropenem 4-Macrolides include: - Rifampicin
&aztreonam)
- Erythromycin 4-Nitroimidazoles
- Azithromycin include:

- Metronidazole

33 | P a g e
 Notes:

Cell wall synthesis:

-penicillins are a large group of antibiotics that include Aminopenicillins (ampicillin&

amoxicillin) penicillinase-resistant penicillin's (oxacillin), penicillins with beta-
lactamase inhibitors (amoxicillin\clavulanic acid).

-methicillin is an antibiotic that was originally used to treat Gram-positive infections

but is no longer available for clinical use. It was replaced by other agents (oxacillin).

-cephalosporin's are further classified into 1st,2nd,3rd and 4th generation according to
their antimicrobial properties.

-cefoxitin is a cephalosporin-like antibiotic that is used for the screening of MRSA.

Protein synthesis inhibitors (PSI):

Other PSI compounds include: linezolid and quinupristin-dalfopristin, both of which
are considered treatment options for infections caused by VRSA.

Cell membrane synthesis inhibitors:

These compounds are highly toxic and poorly absorbed therefor, they are usually
used topically and mainly indicated for Gram-negative infections.

Anti-TB drugs

RNA synthesis Mycolic acid

Fatty acids synthesis
inhibitors include: synthesis inhibitors
inhibitors include:
include:
-Rifampicin
-pyrazinamide(PZA)
-Isoniazid(INH)
-Ethambutol(EMB)

Notes: treatment of TB is usually a combination of:

1-rifampicin, INH, PZA and EMB first for the first 2 months (intensive phase)
34 | P a g e
 2-rifampicin and INH for the further 4 months (continuation phase)

3-in case of TB resistance, other antibiotics are used such as

aminoglycosides(streptomycin)and fluoroquinolones e.g.(moxifloxacin)

Antimicrobial susceptibility testing (AST)

Disc susceptibility test (Kirby Bauer method)

Zone of inhibition=diameter(mm)

Antibiotic disc
Intermediate (I)
Mueller Hinton
agar MH Bacterial growth
Sensitive(S)
Resistant ®

Inoculate the plate with 0.5 McFarland bacterial suspension + Add

antibiotic discs incubate (overnight) > bacterial growth.

Interpret reading using standard chart:

Sensitive>drug is likely to work on patient.

Intermediate>drug may work on patient in higher doses OR in

combination with another drug.

Resistant> drug will not work on patient.

Note: McFarland standards are used as a reference to adjust the turbidity of

bacterial suspensions so that the number of bacteria will be within a given range to
standardize microbial testing. An example of such testing is antibiotic susceptibility

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testing by measurement of minimum inhibitory concentration which is routinely
used in medical microbiology and research. If a suspension used is too heavy or too
dilute, an erroneous result (either falsely resistant or falsely susceptible) for any given
anti-microbial agent could occur

Minimum inhibitory concentration (MIC)

-Is the lowest concentration of an antimicrobial agent that inhibits the visible growth
of a microorganism.

-MBC (minimum bacterial concentration) is the lowest concentration of an

antimicrobial agent that kill a microorganism.

-MIC and MBC are measured in micro gram\ml or mg\l

-bactericidal drugs have MBC values close to their MIC.

-bacteriostatic drugs have MBC values much higher than their MIC values.

Broth dilution method:

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 E Test Method:

Place E test strip on plate inoculated with 0.5 McFarland bacterial

suspension

Incubate (overnight)

Zone of inhibition
Bacterial growth

Mueller Hinton agar

E strip

Example: MIC= 0.25

mg\l point of zone and
strip interception

Report MIC value using

standard chart as: S, I OR
R
Note: 0.5MacFarland
=a.5×10^8 CFU \ml

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 )‫صل الل م سلم على سيدنا مح د على آله صحبه ج عين‬
ِ )

.‫م اصبت ف بتوفيق من ه إ خطأ ف ن نفسي قل حيلتي‬

Remember:

Be a great wherever you go, and be proud of yourself and what you
are accomplished so far. Always remember (great things take time).

GOOD LUCK

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