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Basic Principle of Microbiology
-Different Between Eukaryotes & Prokaryotes :-
1-Eukaryotes ] Nucleus [ :This Cells Form (( Animals –Protozoa- Plants – Fungi –Algea-Helminths )) .
- most eukaryotic cells would lyse at temperature extremes (both hot and cold), with
dryness, and with very dilute and diverse energy sources.
2- Prokaryotes ] No Nucleus [ :- This Cells Form (( Bacteria – Blue green AlgeaMycoplasm-Chlamydia-Rickettsia ))
- Use smaller Ribosome ( 70S ribosome ) .
- Bacteria can survive and, grow in hostile environments in which the osmotic
pressure outside the cell is so low.
- Bacteria have evolved their structures and functions to adapt to these conditions.
-Differences Among Prokaryotes :Bacteria can be distinguished from one another by their morphology (size, shape, and
staining characteristics) and metabolic, antigenic, and genetic characteristics.
Although bacteria are difficult to differentiate by size, they do have different shapes.
A spherical bacterium, such as Staphylococcus, is a coccus; a rod-shaped bacterium,
such as Escherichia coli, is a bacillus; and the snakelike treponeme is a spirillum. In
addition, Nocardia and Actinomyces species have branched filamentous appearances
similar to those of fungi. Some bacteria form aggregates such as the grapelike clusters
of Staphylococcus aureus or the diplococcus (two cells together) observed in
Streptococcus or Neisseria species.
- Bacterial Morphology Shapes :-
- Gram Stain :is a powerful, easy test that allows clinicians to distinguish between the two major
classes of bacteria. Bacteria that are heat fixed or otherwise dried onto a slide are
stained with crystal violet a stain that is precipitated with iodine, and then the
removed by washing with the acetone-based decolorizer and water. A red
counterstain, safranin, is added. This process takes less than 10 minutes.
-Gram Stain Procedures :-
-Result Of Gram Stain :-
1- Gram-positive bacteria :, Color is purple, Because They Have Thick
Peptidoglycan layers .
2- Gram-negative bacteria:- Color is Red, Because They Have Thin Peptidoglycan
layers .
- There are Bacteria cannot be classified by Gram stain Like mycobacteria, which
are distinguished with the acid-fast stain, and mycoplasmas, which have no
peptidoglycan.
Gram Positive Bacteria
Gram-negative bacteria
Cell Wall :- Thick Peptidoglycan
Layers + Tichoic acid + Lipotichoic
acid .
Cell Wall :- Thin Peptidoglycan Layers
+ Lipopolysacchraide ( LPS ) +
Phospholipid + Protein
- Function Of Cell Wall :1- Protect the internal parts of the cell
2- Give The shape of bacteria .
3- Prevent bacteria cell from lysis .
- functions of plasma membrane :1- selective barrier which material enter and exit the cell (selective permeability ).
2- important in break down of nutrient and production of energy .
-Spores :- Found in some gram ve+ bacteria (( e.g bacillus anthracis , clostridium ))
are spore forming , but never gram ve- bacteria .
- spore be within a cell , and is a characteristic of the bacteria and can help in
identification of the bacterium.
- bacterial spores are so resistant to environmental factors .
-Endospore : when essential nutrient or water is unavailable , some gram positive
bacteria like clostridium and bacillus form endospore .
Function of endospore :
1-resistance dehydration and heat .
-Function of ribosome: is synthesis of protein .
- External structures of bacteria :1- Capsules :- Loose polysaccharide or protein layers surround the bacteria .
- it is major virulence factor .
- vi , k antigen found in capsule .
*
function of capsule:
protect bacteria from antibodies and phagocytosis
2- Flagella :- Provides motility for bacteria (( Motor Protein )) .
- it is allow cell to swim ((chemotaxis ))
- chemotaxis is :- toward food and away from poisons .
- H antigen is found in flagella .
The 4 arrangement of flagella :1- monotrichous :- single polar flagella .
2- amphitrichous :- single flagellum at both ends
3- lophotrichous :- two or more flagella at one or both ends .
4- peritrichous :- flagella distributed all over the cell .
5- Atrichous :- without flagella
3- Fimbriae (( Pili )) :- it is promote adherence (( Attachment )) to other bacteria or
the host .
- pili smaller than flagella in diameter . but shorter and thiner than Flagella.
- adherence factor (( Adhesin )) is pili important virulence factor for E.coli .
- O antigen is found in Pili .
-Different between capsule and slime layer :
-capsule : it is organized and firmly attached to cell wall .
- slime layer or(glycocalyx) : it is unorganized and loosely attached to cell wall .
- Biochemical Metabolism and Growth :obligate anaerobes :- bacteria cannot grow in the presence of oxygen.
obligate aerobes :- bacteria require the presence of oxygen for growth .
facultative anaerobes.:- bacteria grow in either the presence or the absence of
oxygen. .
autotrophs (lithotrophs) :- can rely entirely on inorganic chemicals for their energy
and source of carbon (CO
2
).
heterotrophs (organotrophs):- bacteria and animal cells that require organic carbon
sources .
Photosynthetic bacteria :- bacteria which contain Chlorophyll .
- Population dynamics :Lag phase :- adapt with environment in media without growing
Log phase :- the bacteria will grow and divide with doubling time ( multiply ) .
Stationary phase :- stop growing .
Decline :- death increased and stop growing.
Summary of various types of microscopes :1- Brightfield (( light )) microscope :-this microscope stained by Gram stain.
the basic components of this microscope consist of :a- light source (( to illuminate the specimens on the stage )) .
b- condenser (( used to focus the light )) .
c- two lenses to magnify image (( objectives + ocular )) .
- there are 3 different objective lenses are commonly used .:1- low power(( 10 fold magnification )), used to scan specimens .
2- high dry (( 40 fold )), used to look large microbes(( parasites – fungi ))
3- oil immersion (( 100 fold )) . used to observed(( bacteria + yeast )) and
morphologic details of larger organisms.
- the best brightfield microscope have resolving power approximately 0.2um .
2- darkfield microscope :- the same lenses used in brightfield microscope are used in
darkfield microscope .however special condenser is used in this microscope.
- resolving power is 0.02um . this allow to see extremely thin bacteria such as
(( Treponema pallidum : etiologic agent of syphilis ))
3- phase – constrast microscope :- this microscope enables the internal details of
microbe to be examined .
The annular rings use in condenser and the objective lenses .
This microscope give three –dimensional image ((3D)) .
4- Fluorescence Microscope :- this microscope use fluorescent dyes ((stain)).
The microscope use high pressure mercury halogen or xenon vapor .
5- Electron Microscope :- unlike other forms of microscope , magnetic coils ((rather
than lenses )) this microscope used beam of electron instead((‫ )) الد ب ن م‬of light .
Samples are usually stained by metal ions to create contrast .
There are two types of electron microscope:
a- transmission electron microscope : in which electrons pass directly through the
specimen , magnification of it is ((10000 – 100000 X)) and image in this type
not give ((3D)) picture
b- scanning electron microscope : magnify ((1000-10000X)) , the image Is ((3D))
General Information On Microbiology :* Microorganism are divided into three groups on the basis of temperature :1- psycrophiles (cold loving microbes (15-20 C) ) .
2- mesophiles (moderate temperature loving microbes (25-40 C) ).
3- thermophiles (heat loving microbes( 50-60 C) ).
* Microorganism are divided into three groups on the basis of PH :*-Most bacteria grow Best on PH 6.5-7.5 .
1-acidophilic (grow in PH below 7 and can grow on PH 1 ) .. ‫اي ري ت كب ةي ضماح‬
2- Neutrophilic ( grow in PH 7.2-7.4 ) ..‫اي ري ت كب ةل دت عم‬
3-basophilic (grow in PH above 7)
.. ‫اي ري ت كب ةدعاق‬
*Fungi grow between PH 5-6 .
halophiles (grow at high salt , 30% salt )
Acid Fast Stain Procedures :- (used to identify organisms that have waxy
material in their cell wall.)
1- carbol fuchsin.
2- acid-alcohol decolorizing.
3- Counterstain with methylene blue.
Staining using in microbiology :1- Gram stain :- (( most common ))
2- Ziehl-neelsen stain (( acid fast stain ))
3- Wright – giemsa stain
4- India ink
5- Iron hematoxylen stain
6- Auramin – rhodamine stain
-There are different method for staining in microbiology showin in this table:
Staining Method
Use of stain
1- Direct Examination :A wet mount method
A- 10% KOH ( potassium hydroxide )
This method doing by adding water or
saline ( wet mount ) mix with KOH or
lactophenol cotton blue.
Facilitate detection of fungi when added
to lactophenol cotton blue. and dissolve
bacteria.
B- India Ink
Used to detect capsule ssorrounding the
organisms shuch as yeast, Cryptococcus
neoformans.(fungi) .
C- Iodine
Used to different between ameba and
WBC.
2- Differential Stains :A- Gram stain
Most commonly used stain in
microbiology used to different between
gram + from gram B- Iron Hematoxyline Stain
Used for detection fecal protozoa.
C- Methenamine silver stain
Used in histology lab .
D- Trichrome stain
Used for detection fecal protozoa.
E- Wright-Giemsa stain
Used to detect blood parasites
( Chlamydia).
3- Acid fast Stain :A- ziehl-Neelsen stain
-It is the oldest method of acid fast stain .
- used to stain mycobacteria and other
acid fast bacteria ( Heat acid fast stain )
B- Kinyoun stain
Cold acid fast stain ( Not require heating)
C- Auramine-rhodamine stain
Same principle as other acid-fast stains,
except that fluorescent dyes are used for
primary stain .
4- Fluorescent Stains :A- Acridine orange stain
B- Auramine-rhodamine stain
C- Calcofluor white stain
-Used for detection of bacteria and fungi.
-Same as acid-fast stains.
- Used to detect fungi , some lab replaced
KOH stain with this stain.
*
Mycoplasm
: is microorganisms (bacteria) that lack cell wall.
*Mycoplasm
can not be identified by Gram stain.
*Bacteria cells divided by
binary fission
.
*Bacteria exchange genetic information carried by plasmid.
* substanse use in catalyse reaction is H2O2 .
Citrate test :detects the ability of an organism to use citrate as source of energy
.
If the organism has the ability to use citrate, the medium changes its color from green
to blue.
Examples:

Escherichia coli: Negative

Klebsiella pneumoniae: Positive

Frateuria Aurantia: Positive
Indole test:Indole-Positive Bacteria :- E.coli , Haemophilus influenzae, Proteus sp ,
Plesiomonas shigelloides, Streptococcus faecalis, and Vibrio sp.
Indole-Negative Bacteria :- Actinobacillus spp ,most Haemophilus sp., most
Klebsiella sp., Neisseria sp Proteus mirabilis, Pseudomonas sp., Salmonella sp
Yersinia sp. most Bacillus sp., Bordtella sp., Enterobacter sp.,
CULTURE MEDIA :- types of media :-
1- Nutrient agar
: Use for growth all species of bacteria is not have
special environment .
Type of nutrient media :
asimple (basic ) media
:- this media allow growth of all species of bacteria .
because this media contain most bacterial requirement for growth . this media nonselective media .
b- Enriched ( complex ) media :
Some bacteria are fastidious and their growth
requires the presence of highly nutritive .
-Type of enriched media :
hemolytic action
Use for identify bacteria by their
blood agar :
1
2- chocolate agar : It is heated blood agar the temp raised to 100 ºC . and it selective
And inhibit other bacteria .
emophilus group.
Neisseria , Ha
media use for growth
(diphtheriaa bacilli )) .
clostridium diphtheriaa(
use for growth
Loffler serum :
3
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------This media contain substance that inhibit all
selective media :
2
bacteria but allow growth a few type of bacteria .
Types of selective media :A- MacConkey : the most common selective media,
That support the growth of most gram –ve rods espically (Enterobacteriaceae spp) but
inhibits growth gram +ve organisms and some fastidious gram –ve bacteria.
losis (TB).
mycobacterium tubercu
Use for growth
J):
Lowenstein Jensen (L
B
, inhibits all normal flora of upper
diphtheria
Use for growth
Blood tellurite :
C
respiratory tract
N. gonorrhoea
:many special growth factors by
Mattin
–
Thayer
D
. and
onella
Shigella and Salm
use for growth
Desoxycholate Citrate agar (DCA):
E
Inhibits growth intestinal flora.
-this media also differential and selective media for shigella & salmonella
((enterbacteriaces)).
(yellow colonies).
Vibrio cholerae
: use for
TCBS
F
a use for growth
Selective medi
salmonella agar ( SS AGAR ) :
shigella
G
, and inhibt other types of bacteria
salmonella and shigella
a- salmonella (colorless with black center due to produce H2S )
b- Shigella (colorless with no change in center )
.
and
staph spp ,
or growth
selective media use f
mannitol salt agar ( MSA ) :
H
inhibit growth all other types of bacteria . because contain high concentration of salt (
Nacl 7.5% )
(( General
fungi .
selective media for growth of
saburod dextrose agar ( SDA) :
I
media in mycology ))such as ((
Aspergillus flavus, Candida albicans
)) , and inibit growth
of all bacteria spp . because their PH acidic ( ph Below 7 )
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
3- Differential Media :media distinguish one microorganism
type from another growing on the same media .
Types of differential media:
1- MacConkey : different media to differentiate between :a- lactose fermenter ( red
to pink
) e.g : E.coli , Klebsilla , Enterobacter
b- Non lactose fermenter ( colorless ) e.g proteus , salmonella , shigella .
this media contain bile salts + crystal violet that make it allow growth of gram
negative bacteria and inhibit growth of gram positive bacteria .
2-Blood agar plate (BA) :- different media to differentiate between :Streptococcus pneumoniae : Good growth, Alpha - hemolysis
Streptococcus pyogenes : Good growth, Beta-hemolysis
3- Cystine lactose electrolyte deficient ( CLED): different media to differentiate
between :a- lactoe fermenter ( yellow ) e.g E.coli .
b- non lactose fermenter ( colorless ) e.g acintobacter .
this media
Use for urine culture.
4- Triple sugar iron (TSI) agar:
different media to differentiate between :, pseudomonas, E.coli, proteus mirabilia
5- Xylose lysine desoxycholate (XLD) : different media to differentiate between :a- salmonella ( pink color with black center due to produce H2S )
b- Shigella ( pink color with no change in center )
this media
Use for stool culture