Practical Microbiology احياء مجهرية عملي pdf - عملي

Mustafa Hatim Kadhim

Baghdad University

Al-kindy college of medicine

Third Stage

2013 - 2014

List of contents

Lab
number

Lab name

Page
number

Unit 1: General Microbiology (3 – 14)

Sterilization & Disinfection

4 - 5

Culture Media

Preparation of Smear

7 - 8

4+5

Sample

9 - 11

Molecular diagnostics (Polymerase chain
reaction)

12 - 14

Unit 2: Immunology (15 – 21)

1+2+3

Practical Immunology

16 - 21

Unit 3: Bacteriology (22 – 54)



Gram Positive Bacteria (23 – 38)

Staphylococcus + Streptococcus

24 - 28

Bacillus & Neisseria

29 - 32

Clostridium & Mycobacterium

33 - 35

Corynebacteria

36 - 38



Gram Negative Bacteria (39 – 54)

1+2+3

Enteric Gram Negative rods

40 - 47

Vibrio & Heamophilus

48 - 49

Brucella, Campylobacter &
Helicobacter

50 - 51

spirochetes & Chromagar

52 - 54

Unit 4: Mycology (55 – 63)

1+2

Practical Mycology

56 - 63

Unit 5: Virology (64 – 69)

1+2

Laboratory Diagnosis of Viruses

65 - 69

Unit 1: General Microbiology

Lab1 – Sterilization & Disinfection

Instructions in the lab

1) Lab coat should be worn before the entry to the lab over

street clothes and remove before leaving

2) In typical way, laboratory workers should wear goggles,

mask and gloves.

3) Do not eat, drink or smoke inside the lab, these should be

done in isolated room or rest room.

4) Do not bite the nail or put the pen into the mouth.
5) Do not mouth pipette, rubber teat or mechanical pipette

should be used.

6) Assume that patients are infectious for HIV or blood

borne disease.

7) Wash the hands thoroughly after removing the gloves or if

there is any contamination



Sterilization: The processes by which all forms of

microbial life, including vegetative and spore forming
bacteria are destroyed or killed.



Disinfection: The processes that result in the destruction

of only the vegetative forms of microbial life (pathogenic
organisms) but not spores

Sterilization

Physical methods

Heat

Is most practical and dependable method of sterilization,
this method depend on the effective oxidation of
microorganisms by carbonization or coagulation of the
protoplasm.

Dry Heat:

E.g. heating of platinum loop, points of forceps, needles
and mouth of culture tubes ect.



Hot air oven:

this type requires longer exposure times

(1.5-3 Hours) and the temperature is higher than moist
heat (160-180 C) .Hot air oven is the best method for

sterilizing dry glass wares, oil petrolatum, liquid paraffin
powders,…

 The oven should be set to 160 C for 2 hours, 170 C for 1

hour, 180 C for 30 Min.

b) Moist heat

1) At a temp. below 100 C:

For example pasteurization of milk, the temp. employed either
63-65 c for 30 min. or 71 c for at least 15 seconds. This will
kill all non-spore forming pathogens such as Mycobacterium
tuberculosis, Brucella abortus and various Salmonellae, so that
this method can be used to kill food pathogen

2) At a temperature of 100 c

a) Boiling at 100 c for 5-10 min.

This is sufficient to kill all non-sporing & many but not all
spore-forming microorganisms, used to sterile water.

b) Steaming at 100 c .for example:

Tyndallization which involves steaming for 30 min. on
each 3 successive days.
After heating, the medium is incubated to allow spores
to germinate and vegetative organisms to grow which
will be killed by the second steaming; the 3

heating is

a safety precaution .used for culture media containing
sugar or gelatin.

3) At a temperature above 100c



Steam under increased pressure, this is called

(autoclaving). This is the usual method of sterilization
bacteriological media ,surgical instruments, towels,
dressing ,gloves ,………ect



The 2 common sterilization temp. either

a) 121c for 15 min for sterilizing items such as media,

liquid and medical instruments.

b) Infectious waste such as unused portion of patients’

specimens, patients’ culture, sharp disposable sharp
instruments such as scalpels, needles sterilized at 132
C for 30-60 Min.

Radiation:

Act on nucleic acid, used in commercial purpose to sterile
high quantity of pre packed material

a) Direct sun light

kills vegetative organisms rapidly, but

spores are much more resistant.

b) Ultra violet light

: is commonly used to sterilize the air in

the surfaces (used in Bacteriological hood)

c) Ionizing radiation

includes high speed electrons .X-rays

.It has effect on under surfaces. This method is used for
sterilizing disposal such as gloves catheter, plastic syringe
and Petri dishes.

Unit 1: General Microbiology

d) Infrared radiation

: by electrically heat element, a

temp180 C can be attained .used for glassware.

Filtration

To render fluids, including bacterial culture, free from
bacteria by passing them through special filters e.g.seitz
filter. This method is used in sterilizing liquids that
would be damaged by heat such as serum, vaccines
,antibiotic solutions, sugars, toxins ………etc.

Chemical methods

It act on lipid contents of the cell membrane denaturation
of protein, and on nucleic acid, it either kill or stop the
growing of microorganism.

a) Potent: disinfectant:

It is toxic and corrosive for living tissue, it’s used for non-
living

1) Phenol group

: contain benzene ring e.g.bathrooms,

hospital, floor ……ect.

2) Chlorine

is most often used in form of sodium

hypochloride (house hold bleach) in dilution (1:10)
treatment of swimming pools etc.

3) Strong alkaline and acids

e.g. NAOH

Used for treating sputum for detecting TB and decrease
viscosity

b) Mild: Antiseptics:

It is less toxic and can be applied to living tissue like
skin. eg.

1) Detole
2) Chlorhexiden (Hibitane):

very good as skin antiseptics

used to treat surgical wounds because it work on both
gram-positive and gram-negative bacteria

3) Iodine:

used in surgery to sterile skin pre-operation.

4) H2O2(hydrogen peroxide )

can be used for sterilization

of deep wound or gangrene infected by anaerobic bacteria

5) Soap and detergent.
6) Alcohol

at conc.70% because it can penetrate tissue easily

at this concentration better than 99%(absolute)

Unit 1: General Microbiology

Lab 2 - Culture Media

Unit 1: General Microbiology

Lab 3 - Preparation of Smear

Unit 1: General Microbiology

Lab 4+5 - Sample

Unit 1: General Microbiology

Lab 6 - Molecular diagnostics
(Polymerase chain reaction)

Unit 1: General Microbiology

Unit 2 - Immunology

Lab 1+2+3 – Practical Immunology

Unit 2 - Immunology

Agglutination inhibition

Hemagglutination inhibition (Influenza virus)

Indirect immune
fluorescence test

Unit 2 - Immunology

Unit 3 - Bacteriology

Lab 1 – Staphylococcus + Streptococcus

Staphylococcus

Unit 3 - Bacteriology

The streptococci

Unit 3 - Bacteriology

Lab 2 – Bacillus & Neisseria

Bacillus

Neisseria

Unit 3 - Bacteriology

Lab 3 - Clostridium & Mycobacterium

Clostridium species

Unit 3 - Bacteriology

Mycobacteria (Mycobacterium spp.)

Unit 3 - Bacteriology

Lab 4 – Corynebacteria

7-10 colonies of C.
duphtheriae on 5%
sheep blood agar.
Corynebacterium
diphtheriae grow
well on 5% sheep
blood agar as
whitish, opaque
colonies

Unit 3 - Bacteriology

Lab 1+2+3 – Enteric Gram Negative rods

Unit 3 - Bacteriology

Lab 4 - Vibrio & Heamophilus

Vibrio

Unit 3 - Bacteriology

Haemophilus

A: Identification of H.influenzae by X & V strips
B: H.influenzae satelliting around staphylococcus aureus

Unit 3 - Bacteriology

Lab 5 – Brucella, Campylobacter &
Helicobacter

Brucella

Campylobacter

Unit 3 - Bacteriology

Helicobacter

Unit 3 - Bacteriology

Lab 6 – spirochetes & Chromagar

Unit 3 - Bacteriology

Chromagar

Unit 3 - Bacteriology

Unit 4 - Mycology

Lab 1+2 - Practical Mycology

Medical Mycology is the science that deals with
medically important fungi (fungus), fungi belong to the
kingdom Fungi.

General features of the fungi:



Fungi seen in the clinical laboratory can generally be

separated into two groups based on the appearance of the
colonies formed. The yeasts are produced as moist,
creamy opaque or pasty colonies on media, while the
filamentous fungi or molds produce a fluffy cottony
woody or powdery colony.



Several pathogenic spp. of fungi that exhibit either a yeast

or filamentous appearance are called dimorphic.

Yeasts

Yeasts are unicellular structures that are round to oval in
shape. The microscopical morphology features usually
similar among genera and difficult to differentiate
however, the size the presence or absence of capsule or
narrow necked budding are features that can be helpful to
differentiate between Cryptcoccus spp. and from Candida
spp.

Molds

The basic structural units of the molds are tube like
projection known as hyphae , as the hyphae grow, they
become intertwined to form loose network called
mycelium, which penetrate the substrate from which the
molds obtain the necessary nutrients for growth, this is
called vegetative mycelium. The portion extend above the
substrate surface, is known as aerial mycelium that are
responsible for the fluffy filamentous nature of the mold.

Three types of hyphae exist in medically important
fungi these include

1) The sparsely septate (a septate hyphae) ,an example of

zygomycetes

2) The dark pigmented septate hyphae an example of

dermatiaceous fungi.

Unit 4 - Mycology

3) The septate non pigmented hyphae an example of hyaline

molds.

Unit 4 - Mycology

Laboratory diagnosis of fungal infection

The diagnosis of fungal infection depends entirely on the
selection and collection of an appropriate clinical
specimen for culture. The proper collection of the
specimen and their rapid transport to the clinical
laboratory is of a major importance for recovery of fungi.

 Type of specimen



Respiratory secretion
Sputum, induced sputum, bronchial washing and
transtrachial aspiration are the most commonly submitted
specimen for fungal culture.



Cerebrospinal fluid (CSF)
CSF collected for culture should be filtered through 0.45
Mm membrane filters attach to a sterile syringe. After
filtration, the filter is removed and is placed onto the
surface of an appropriate culture media & examine daily.



Blood
The concentration blood is incubated onto the surface
appropriate medium and most fungi are detected within
the first 4 days except Histoplasma capsulatum may
require approximately 10-14 days for recovery.



Hair, Skin and Nail Scrapings
o These specimens are usually submitted for

dermatophytes culture and may be contaminated with
bacteria and rapidly growing fungi sample collected
from lesions may be obtained by scraping the skin or
nail with a scraped blade or microscopical slide

o Infected hairs are removed by plucking them with

forceps .Clipping may be taken from the distal border
of the nail with scissor or nail clapper when the
dystrophy extend to the distal end of the nail.

o These specimens should be placed in sterile pertridish

and they should not be refrigerated. The agar contains
chloramphenicol and cyclohexamide for recovery of
dermatophytes, culture should be incubated at 30

for 30 days. before being reported as negative.



Urine
All urine samples should be centrifuged and the sediment
cultured by loop because urine is often contaminated with
G –ve bacilli, it is necessary to use media containing
antibacterial and antifungal agents.



Tissues, Bone marrow and Sterile body fluids
All tissues should be processed before culturing by
mincing or grinding after processing, at least 1ml of the
specimen should be cultured as soon as possible on
appropriate media and incubated at 30

C for 30 days.

Bone marrow may be placed directly to the appropriate
medium, sterile body fluids should be concentrated by

centrifugation before culturing and at least 1ml of the
specimen should be cultured onto the surface of an
appropriate medium.

 Culture media and Incubation requirements



For optimal recovery a battery media should be used and

the following points recommended:

1) Media with and without blood enrichment 5% sheep

RBCs blood.

2) Media with and without cycloheximide 0.5 mg/ml.
3) All media should contain antibacterial agent except

specimen sterile body fluids



Sabauroud’s dextrose agar is the most common media
used for recovery of pathogenic fungi. Agar plates or
screw capped agar tubes are satisfactory for the recovery
of fungi but plates are preferred since they provide better
aeriation of culturing, a large surface area for better
isolation of colonies and easily handling for making
microscopical examination.

 Direct microscopical examination of clinical specimen

A. Wet mount preparation: This method done by take a

portion of colony from point intermediate between center
and periphery place in a slide and a drop of lactophenol
cotton blue stain is added and then place a cover slide
press firmly then examine microscopically.

B. Gram stain is used for detection of most of fungi that

stain well however others such as Cryptococcus stain
weakly.

C. Calcofluor white is florescent stain for detection of fungi

rapidly because of bright fluorescence and this can be
mixed with KOH.

D. Indian Ink is important of Cryptococcus neoformans to

demonstrate the capsule in CSF when positive in CSF
diagnosis of meningitis (50%of case meningitis give
positive results).

Unit 4 - Mycology

E. E-Potassium hydroxide KOH preparation 10-20% used

for cleaning of specimen tissue to make fungi more
reading visible by digest protein and clear keratinized
tissue. If takes 5min. for clearing. If clearing is not
complete add another 5-10mins. This is a rapid method
for detection of fungi element.

General morphologic features of molds

 Conidia
 Arthroconidia

 Chlamydioconidia

Fungi may produce conidia of two sizes: microconidia:
small unicellular, round elliptic in shape or macroconidia:
large, multiseptated, club or spindle shaped. Microconidia
may be formed on the side of the hyphae on the end of the
hyphae but macroconidia or usually bare on a
conidiophore and may be smooth or roughly walled.

 Sporangium:

Sporangiospores: they are spores produced within the
sporangium and released by rupture of the sporangial wall.

 Hyaline septate molds



Cutaneous infection (Dermatophytes)

Dermatophytes includes genus Trichophyton that capable
of invading the hair nail and skin. Genus microsporum
involve dry hair and skin, The genus Epidermophyton
involve the skin and nail.
o Direct detection
o Culture and Identification



Trichophyton mentagrophytes

o Colonial morphology White to pinkish granular and fluffy

slow growing colonies.

o Microscopical Identification

Many round microconidia most commonly borne on
grape like clusters or laterally along the hyphae:
macroconidia are thin walled, smooth, club shaped and
multiseptate.

Unit 4 - Mycology



Trichophyton rubrum

o Colonial morphology

Colonial types vary from white to pink granules

o Microsopical Identification

Microconidia is usually teardrop shaped or club shaped
most commonly borne a long sides of hyphae,
macroconidia is usually absent but when present are thin
or smooth walled.



Microsporium canis

o Colonial morphology

Colonies of Microsporium canis grow rapidly; they are
granular or fluffy.

o Microsopical Identification

Thick walled, spindle shaped, large multisepate rough
walled macroconidia, some with a curved tip,
microconidia rarely seen.



Epidermophyton

Epidermophyton floccosum

o Colonial morphology

Colony grows slowly and the growth appears khaki green
in the center and tends to folded, the periphery is yellow.

o Microsopical Identification

Macroconidia large ,smooth walled numerous,
multiseptate, they are round at top and are borne singly in
conidiophore or in group of two or three. Microconidia
are absence.

 Hyaline, septate molds (opportunistic mycosis)



Aspergillus fumigatus

o Is the most common spp. recovered from immune

compromised patients, and it is the most common spp.
seen in clinical lab.

o Aspergillus fumigatus is a rapidly growing mold 2-6 days

that produce a fluffy to granular, white to blue green
colony.

Unit 4 - Mycology

o Microscopically is characterized by the presence of

septate hyphae branching with short or long conidiophore.
The tip of conidiophore expand into a large, dome shaped
vesicle that has bottle- shaped covering the upper half of
its surface.



Aspergillus niger

o Produce mature colonies within 2-6 days. Growth begins

as yellow colonies then develop a black, dotted surface as
conidia is produced.

o Microscopically

Aspergillus niger shows septate hyphae, long
conidiophore that support spherical vesicles.



Penicillium

o Penicillium is one of the most common organisms

recovered by clinical lab. Colonies of Penicillium are
green, blue green, pink or white .

o Microscopically the hyphae are hyaline septate and

produce brush like conidiophores (penicillens).

 Hyaline septate dimorphic molds



Blastomyces dermatitidis

o Direct detection

B. dermatitides appear as large, spherical, thick walled
yeast cell usually with a single bud that is connected to
the parent cell by broad base

o Culture and Identification

The mold form develops as well as waxy appearing
colonies that become off-white to white color.
Microscopically the hyphae of the mold form are septate,
and single teardrop conidia on long to short conidiophore.
When incubated at 37

C colonies of the yeast form

develop in 7 days and appear waxy, and cream to tan in
color. Microscopically large, thick walled yeast cell with
bud attached by a broad base.

o Coccidiodes immitis produce spherules which contain non

budding endospore microscopically.

Unit 4 - Mycology

o Histoplasma capsulatum produce small, budding yeast

cell within the macrophage seen microscpically.

o Sporothrix schenkii in direct microscope show small

round to oval to cigar shaped yeast cell.

Yeast

 Ex. Candidia



Direct examination

The direct examination microscopically of clinical
specimen containing Candidia reveal budding yeast cell
(Blastoconidia) and/ or Pseudohyphae showing regular
point of constriction or true hyphae.

The Blastoconidia, hyphae. Pseudohyuphae are strongly
Grams +ve



Culture and Identification

Colonies of Candidia are creamy, moist, and opaque with
sweaty odor.
Candida albicans may be identified by germ tube
production or production of chlamydospores on corn
meal agar .

o Germ tube method

Suspend a very small inoculum of yeast cell from isolate
colony in 0.5 c.c. of sheep serum then incubate at 37

for up to 3hr. often incubation a drop of suspension is
removed and place on shade and examine under low
power for the presence of germ tube which is appear as
appendage extended from mother cell that is half the
width and 3-4 times the length of the yeast cell.

Unit 4 - Mycology

 Cryptococcus

Ex. Cryptococcus neoformans



Direct examination

Indian Ink preparation in most widely used method for
rapid detection of Cryptococcus neoformans in clinical
specimen ,Cryptococcus neoformans appear as spherical,
single or multiple budding thick walled yeast like
organism surrounded by wide retractile capsule.



Culture and identification

Colonies of Cryptococcus neoformans usually appear on
culture media within 2-5 days and begin as smooth, white
to tan colony that may become mucoid cream to brown in
color.

Unit 5 - Virology

Lab 1+2 - Laboratory Diagnosis of
Viruses

Unit 5 - Virology

106

107

Unit 5 - Virology