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 Algea-M ycoplasm-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 conditi ons.

N.B: -Differences Among Prokaryotes :Bacteria can be distinguished from one another by their morphology (size, shape, and staining characteristics) and metabolic, antigenic, and genetic characteris tics. Although bacteria are difficult to differentiate by size, they do have dif ferent shapes. A spherical bacterium, such as Staphylococcus, is a coccus; a rod -shaped bacterium, such as Escherichia coli, is a bacillus; and the snakelike tr eponeme is a spirillum. In addition, Nocardia and Actinomyces species have branc hed filamentous appearances similar to those of fungi. Some bacteria form aggreg ates 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 m ajor 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 Peptidogly can layers . 2- Gram-negative bacteria:- Color is Red, Because They Have Thin Peptidoglycan l ayers . - There are Bacteria cannot be classified by Gram stain Like mycobacteria, whi ch are distinguished with the acid-fast stain, and mycoplasmas, which have no pe ptidoglycan. Gram Positive Bacteria Gram-negative bacteria Cell Wall :- Thick Peptidoglycan Layers + Tichoic acid + Lipotichoic acid . Cell Wall :- Thin Peptidoglycan Layers + Lipopolysacchraide ( LPS ) + Ph ospholipid + 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 permeabil ity ). 2- important in break down of nutrient and production of energy .

-Spores :- Found in some gram ve+ bacteria (( e.g bacillus anthracis airobic, cl ostridium anairobic)) 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 positi ve 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: ( protiction ) 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 bact eria 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 wa ll .

- 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 th urce of carbon (CO2) . heterotrophs (organotrophs):- bacteria and animal cells that require organic car bon 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 ) c ell # will increase . 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 mor phologic 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 us ed 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 contrast 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 ((ra ther than lenses )) this microscope used beam of electron instead(( )) of light . Samples are usually stained by metal ions to create contrast . There are two types of electron microscope: atransmission electron microscope : in which electrons pass directly thro ugh the specimen , magnification of it is ((10,000 100,000 X)) and image in this type not give ((3D)) picture bscanning electron microscope : magnify ((1,000-10,000X)) , the image Is ((3D))

General Information On Microbiology :* Microorganism are divided into three groups on the basis of temperature :1psycrophiles (cold loving microbes (15-20 C) ) . 2mesophiles (moderate temperature loving microbes (25-40 C) ). 3thermophiles (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 ) 2- Neutrophilic ( grow in PH 7.2-7.4 ) .. 3-basophilic (grow in PH above 7) .. *Fungi grow between PH 5-6 . halophiles (grow at high salt , 30% salt ) Acid Fast Stain Procedures :- (used to identify organisms that have waxy materi al in their cell wall.) 1- carbol fuchsin. 2- acid-alcohol decolorizing. 3- Counterstain with methylene blue. Staining using in microbiology :1Gram stain :- (( most common )) 2Ziehl-neelsen stain (( acid fast stain )) 3Wright giemsa stain 4India ink 5Iron hematoxylen stain 6Auramin rhodamine stain

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-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 lactop henol cotton blue. Facilitate detection of fungi when added to lactophenol cotton blue. and dissolv e 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 g ram B- Iron Hematoxyline Stain Used for detection fecal protozoa. in stool C- Methenamine silver stain Used in histology lab . D- Trichrome stain Used for detection fecal protozoa. in stool 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 o ther 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. Without 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 . * IgA(important in mucosal infections)

Citrate test :- detects the ability of an organism to use citrate as source of e nergy. If the organism has the ability to use citrate, the medium changes its color fro m green to blue. Examples: Escherichia coli: Negative Klebsiella pneumoniae: Positive Frateuria Aurantia: Positive Indole test:Indole-Positive Bacteria :- E.coli , Haemophilus influenzae, Proteus sp , Plesi omonas shigelloides, Streptococcus faecalis, and Vibrio sp. Indole-Negative Bacteria :- Actinobacillus spp ,most Haemophilus sp., most Kleb siella sp., Neisseria sp Proteus mirabilis, Pseudomonas sp., Salmonella sp Yersi nia 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 en vironment . Type of nutrient media : a-simple (basic ) media :- this media allow growth of all species of bacteria . because this media contain most bacterial requirement for growth . this media no n- selective media . b- Enriched ( complex ) media : Some bacteria are fastidious and their growth re quires the presence of highly nutritive . -Type of enriched media : 1- blood agar : Use for identify bacteria by their hemolytic action 2- chocolate agar : It is heated blood agar the temp raised to 100 C . and it sel ective media use for growth Neisseria , Haemophilus group. And inhibit other b acteria . selective media inhibit growth of other 3- Loffler serum : use for growth clostridium diphtheriaa((diphtheriaa bacilli ) ) . -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2- selective media : This media contain substance that inhibit all bacteria bu t allow growth a few type of bacteria . Types of selective media :- ( 9 )

A-) MacConkey : the most common selective media, That support the growth of most gram ve rods espically (Enterobacteriaceae spp) b ut inhibits growth gram +ve organisms and some fastidious gram ve bacteria. B-) Lowenstein Jensen (L-J): Use for growth mycobacterium tuberculosis (TB). C-) Blood tellurite : Use for growth diphtheria , inhibits all normal flora of u pper respiratory tract . D- Thayer Mattin :many special growth factors by N. gonorrhoea E -) Desoxycholate Citrate agar (DCA): use for growth Shigella and Salmonella . and Inhibits growth intestinal flora. -this media also differential and selective media for shigella & salmonella ((en terbacteriaces)). F-) TCBS : use for Vibrio cholerae (yellow colonies). G-)shigella - salmonella agar ( SS AGAR ) :Selective media use for growth salmon ella and shigella , and inhibt other types of bacteria a- salmonella (colorless with black center due to produce H2S ) b- Shigella (colorless with no change in center ) . H-) mannitol salt agar ( MSA ) :- selective media use for growth staph spp , an d inhibit growth all other types of bacteria . because contain high concentratio n of salt ( Nacl 7.5% ) I-) saburod dextrose agar ( SDA) :- selective media for growth of fungi .(( Gene ral media in mycology )) such as (( Aspergillus flavus, Candida albicans)) , a nd inibit growth of all bacteria spp . because their PH acidic ( ph Below 7 ) = mycology -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3- Differential Media :- media distinguish one microorganism type from another g rowing 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 , C itrobacter , Serratia spp , Psudomonase 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 . N.B : macConkey : selective cus allow growth of gram ve and inhibit growth of gra m +ve , differential cus differentiate between lactose & non-lactose . 2-Blood agar plate (BA) :- different media to differentiate between :Streptococcus pneumoniae : Good growth, Alpha hemolysis partial breakdown or he molysis of blood Streptococcus pyogenes : Good growth, Beta-hemolysis complete hemolysis of b lood ( go to mofa9'ala ) 3- Cystine lactose electrolyte deficient ( CLED): different media to differentia te 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 6- shigella salmonella agar ( SS AGAR ) : different media to differentiate betwe en a- salmonella (colorless with black center due to produce H2S ) b- Shigella (colorless with no change in center ) 7- mannitol salt agar ( MSA ) :- different media to differentiate between :Staph spp ( not ferment mannitol ) . Staph aureus ( ferment mannitol with golden color )

8-bile esculine : different media to differentiate between :Strepto spp Streptococcus gamma enterococcus (( E.faecalis )), that produce enzyme form blac k color . This media for stool sample . this media contain Iron (( ferrous )) 9-Eosin Methylene Blue Agar (EMB) different media to differentiate between :Gram-negative enteric bacteria , Bacteria that ferment lactose, especially the c oliform bacterium Escherichia coli from non lctose fermentive like macConkey Escherichia coli : Good growth, green metallic sheen . sheen = lm3an Klebsiella pneumoniae : Good growth, purple colonies, no sheen. Shigella flexneri : Good growth, transparent colonies (lactose negative) --------------------------------------------------------------------------------------------------------------------------*Other type of media: * Mueller Hinton agar :Less nutrient media the best use of this media is for Sensitivity tests .. because it not contain a ny chemical substances . -bordet gangou medium is the best isolation of brodetella retusis Hektoen agar: for isolation Shigella only

General information about media :Blood agar contain:* 1) agar 2)mineral salts 3)tryptone 4)meat extract *Media to become nutritive must contain the :1- source of carbon . 2- source of nitrogen 3- water 4- minerals bordet gengous agar or blood charcoal agar are specific media for Bordetella per tussis . Human is the only natural host for S. typhi & S Paratyphi . By using darkfield microscope may able to detect the motile Vibrio cholera bacteria in stool specimen ( so Darkfild microscope ca n used for syphlis & V.cholera in stool