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Basic microbiology lab requirements : Instruments and materials : 

Basic microbiology lab requirements : Instruments and materials

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Bunsen burner : to work on sterile environment and sterilize wire loops

IMMERSION CEDAR OIL : for oil immersion lens microscopy ON OIL : 

IMMERSION CEDAR OIL : for oil immersion lens microscopy ON OIL

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Wire ( Platinum loops: to cultivate and transfer Mos from specimens or cultures

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Cotton Swab : to obtain clinical specimens e.g. nasal, eye , vaginal , rectal swabs

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Petri dishs ( plastic or glass )

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Plastic syringes : to otain blood & various body fluids

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COLONY COUNTER : to count colonies of Mos obtainrd after culture e.g. urine samples

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Electric centrifuge : to sediment specimens e.g. Urine & do thenafter the assinged test

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CO2 jar ( Candle jar ): to grow microaerophilic Mos e.g. Neisseriae spp. + Hemophilus spp.

Anaerobic jar : to grow anaerobic Nos e.g. Clostridium spp.+ bacteroides spp.: 

Anaerobic jar : to grow anaerobic Nos e.g. Clostridium spp.+ bacteroides spp.

Autoclave : to sterilize culture media & different reagents by moist heat: 

Autoclave : to sterilize culture media & different reagents by moist heat

Seitz filter : to sterelize heat sensitive fluids e.g. serum & antibiotic solutions : 

Seitz filter : to sterelize heat sensitive fluids e.g. serum & antibiotic solutions

Gram,s stain- 4 reagents: 

Gram,s stain- 4 reagents

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Appearance of the Gram positive coccus and Gram negative bacillus at different stages of the gram staining procedure are illustrated below :

TB ZIEHL-NEELSEN KIT – 3 reagents : 

TB ZIEHL-NEELSEN KIT – 3 reagents

Negative stain kit: 

Negative stain kit Contains Nigrosin Solution that do not stain cells but makes them visible against a stained background • Assists in detecting of spiral of MOs • Great for studying cell morphology Used for detection of capsules

Staphylococci spp.( gram stain ) : gram positive cocci ( blue or violet ), arranged in clusters or grapes,: 

Staphylococci spp.( gram stain ) : gram positiv e cocci ( blue or violet ), arranged in clusters or grapes,

Streptococci spp.( gram stain ): gram positive cocci ( blue or violet), arranged in chains: 

Streptococci spp.( gram stain ): gram positive cocci ( blue or violet), arranged in chains

Diplococci( gram stain ): gram positive ( blue or violet ) Strep. Pneumoniae , Strep. viridans , Enterococci spp, arranged in pairs: 

Diplococci ( gram stain ): gram positive ( blue or violet ) Strep. Pneumoniae , Strep. viridans , Enterococci spp , arranged in pairs

Neisseriae spp. ( Gram stain( : gram negative cocci ( red ), arranged in pairs, located intracellular inside pus cells and extracellular: 

Neisseriae spp. ( Gram stain ( : gram negative cocci ( red ), arranged in pairs, located intracellular inside pus cells and extracellular

Gram positive bacilli, Non-spore former ( Gram stain ) : gram positive bacilli ( blue or violet ), arranged in V- or L- shape (Chinese –letter arrangement ). Ex. Corynebacteria spp.: 

Gram positive bacilli, Non-spore former ( Gram stain ) : gram positive bacilli ( blue or violet ), arranged in V- or L- shape (Chinese –letter arrangement ) . Ex. Corynebacteria spp.

Gram positive bacilli, spore former: ( Gram stain) :gram positive bacilla ( blue or violet ), arranged in chains, with empty center indicate the spore : 

Gram positive bacilli, spore former: ( Gram stain) :gram positive bacilla ( blue or violet ), arranged in chains, with empty center indicate the spore

Acid – fast bacilli ( Zheil Neelsen stain ): Mycobacterium spp. : red bacilli, arranged in small groups as a nest. : 

Acid – fast bacilli ( Zheil Neelsen stain ) : Mycobacterium spp. : red bacilli, arranged in small groups as a nest.

Gram negative bacilli ( Gram stain ): gram negative bacilli ( red ), arranged in small groups: 

Gram negative bacilli ( Gram stain ): gram negative bacilli ( red ), arranged in small groups

India ink stain (Negative stain ) : To demonstrate spirochete. Long , thin, spirals , transparent with dark background: 

India ink stain (Negative stain ) : To demonstrate spirochete. Long , thin, spiral s , transparent with dark background

Gram stain : Candida spp. : large oval to round yeast cells stained blue or violet: 

Gram stain : Candida spp. : large oval to round yeast cells stained blue or violet

Candida albicans, pseudohyphae or :germ tube formation i.e.branching as if a mold morphology: ( Gram stain ) : 

Candida albicans , pseudohyphae or : germ tube formation i.e.branching as if a mold morphology: ( Gram stain )

Aspergillus spp.: ( Lactophenol cotton blue stain ) : 

Aspergillus spp. : ( Lactophenol cotton blue stain )

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Aspergillus species produce clear septate hyphae . On the ends of the specialized pieces of hyphae (conidiophores) is a swollen tip (vesicle) On these vesicles, phialides (or sterigma ) are present, and long strands of small spores are borne at the end of the sterigma .

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Penicillium spp. -( Penicillis - Latin for brush) ( Lactophenol cotton blue stain )

Penicillium spp. -(Penicillis - Latin for brush) :( Lactophenol cotton blue stain ) : 

Penicillium spp. -( Penicillis - Latin for brush) : ( Lactophenol cotton blue stain ) The thallus (mycelium) typically consists of a highly branched network of multinucleate, septate , usually colorless hyphae . Many-branched conidiophores sprout on the mycelia, bearing individually constricted conidiospores often are green in color.

Cryptococcus neoformans : India ink staining: 

Cryptococcus neoformans : India ink staining Cryptococcus neoformans by India ink staining ( Negative stain ). Note the wide halo zone representing the capsule . Note too some of this yeast showing budding.

Nutrient broth : 

Nutrient broth Infusion or digest meat broth to which 1% Peptone &0.5% NaCl are added. A good all purpose isolation and maintenance medium. Use the broth for shake culture. In nutrient broth, the bacteria grows in the liquid, and is seen as a soupy substance, not as clearly distinguishable clumps.

Nutrient agar: 

Nutrient agar Infusion or digest meat . It is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. It is useful because it remains solid even at relatively high temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly visible as small colonies

Blood agar: 

Blood agar Nutrient agar Plus 5-10% human or animal blood. Use :  For the isolation, cultivation and detection of hemolytic activity of streptococci, pneumococci and other particular fastidious microorganisms. Control organisms : Streptococcus pneumoniae : Good growth, Alpha - hemolysis Streptococcus pyogenes : Good growth, Beta- hemolysis Incubate: aerobic 35° C, 24 hrs.

Chocolate agar:- Heated blood agar : 

Chocolate agar:- Heated blood agar Use : For the isolation and cultivation of a variety of fastidious MOs. Control organisms : Hemophilus influenza and Neisseria gonorrhoeae : Good growth IncubateCO 2 ,35° C ,24 hrs.

Mac Conkey agar: 

Mac Conkey agar A selective and differential media containing bile salts, lactose & an indicator for acid production . Use : For the selective isolation, cultivation and differentiation of coliforms and enteric pathogens based on the ability to ferment lactose. Lactose– fermening organisms appear as red to pink colonies. Lactose- nonfermenting organisms appear as colorless or transparent colonies Control organisms : Escherichia coli : Pink colonies (lactose fermentation) Proteus mirabilis : Colorless colonies ,no spreading Enterococcus sp. : No growth Incubate : Aerobic, 35° C

Robertson's cooked meat medium: 

Robertson's cooked meat medium Anaerobic medium containing broth& minced meat. is used for cultivation of aerobes and anaerobes, especially pathogenic Clostridia. This can also be used as a maintenance medium for stock cultures.

Loeffler serum slope: 

Loeffler serum slope An enriched medium especially useful for cultivation of diphtheria bacilli producing good growth within 12 hours before other bactereria can grow well.It is made of glucose broth to which sterile inspissated serum is added . It is creamy in colour . This medium can also be used as a transport medium.

Lowenstein Jensen medium: 

Lowenstein Jensen medium Selective medium for growth of tubercle bacillus , contain egg, glycerol & malachite green . Use : For the cultivation and differentiation of Mycobacterium species.

Sabouraud Agar in petri dishes & slopes : 

Sabouraud Agar in petri dishes & slopes Sabouraud , s glucose agar is used for mycological cultures The pH of the medium is important and is set at 5.4-5.8 . Culture medium is kept in test tubes or plates. Antibiotic solutions are mixed in the medium for special fungi if required .

Eosin Methylene Blue ( EMB ): 

Eosin Methylene Blue ( EMB ) Use : For the isolation, cultivation and differentiation of Gram-negative enteric bacteria based on lactose fermentation. Bacteria that ferment lactose, especially the coliform bacterium Escherichia coli , Appear as colonies with green metallic sheen or blue–black to brown color. Bacteria that do not ferment lactose appear as colorless or transparent light purple colonies Control organisms : Escherichia coli : Good growth, green metallic sheen. Klebsiella pneumoniae : Good growth, purple colonies, no sheen. Shigella flexneri : Good growth, transparent colonies (lactose negative) Incubate : Aerobic 35° C, 24 hrs

Antibiotic sensitivity & Factors affecting antibiotic sensisitivit tests : 

Antibiotic sensitivity & Factors affecting antibiotic sensisitivit tests Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics . Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo . Testing for antibiotic

kirby-Bauer method( Disc diffusion ) : 

kirby -Bauer method( Disc diffusion )

SEROLOGY (ANTIGEN- ANTIBODY TESTS) : 

SEROLOGY (ANTIGEN- ANTIBODY TESTS) Serology is a blood test to detect the presence of antibodies against a microorganism. As well as Antigens of various microorganisms. Serology is the scientific study of blood serum and other bodily fluids. In practice, the term usually refers to the diagnostic identification of antibodies or antigens in the serum. Such antibodies are typically formed in response to an infection (against a given microorganism ), against other foreign proteins (in response, for example, to a mismatched blood transfusion ), or to one's own proteins (in instances of autoimmune disease ). Serological tests may be performed for diagnostic purposes when an infection is suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's blood type . Serological tests help to diagnose patients with certain immune deficiencies associated with the lack of antibodies , such as X-linked agammaglobulinemia . In such cases, tests for antibodies will be consistently negative. Serological tests may also be used forensically , generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample). Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva , which have (roughly) similar properties to serum.

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Latex agglutination test

ELEK'S GEL PRECIPITAION TEST: 

ELEK'S GEL PRECIPITAION TEST one of the double immunodiffusion procedures : A rectangular strip of filter paper soaked with diphtheria antitoxin (1000 units /ml ) is placed on the surface of a 20 normal horse serum agar in a Petri dish. Narrow streaks of test Corynebacterium diphtheria strain are made at right angles to the filter paper strip. A positive and a negative control should be put. Plate is incubated at 37 0 c for for 24-28hours. Toxin production by bacterial growth will diffuse in the agar and where it meets the antitoxin at optimal concentration. The line of precipition indicates that the strain is toxigenic No precipitate will form in case of non - toxigenic strain . It also helpful in differentiation between C. diphtheriae from Diphtheroids .

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Radial or single immunodiffusion ( RID )

ELISA: 

ELISA -Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrenemicrotiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. -The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation . -Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic  substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Staphylococci spp.: 

Staphylococci spp.

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TEST Pathogenic Non-Pathogenic Coagulase test +v e -v e Beta- haemolysis on Blood agar +v e -v e Pigment production on BA or NA Golden Yellow White or LemonYellow DNA se test +v e -v e Mannitol fermentation +v e -v e LAB. DIAGNOSIS OF STAPHYLOCOCCI

Coagulase test :slide and tube methods : Staphylococcus aureuse +ve Staph. epidermidis and Staph. Saprophyticus -ve: 

Coagulase test :slide and tube methods : Staphylococcus aureuse + ve Staph. epidermidis and Staph. Saprophyticus - ve

Catalase is responsible for the breakdown of hydrogen peroxide into oxygen and water : Staphylococci +ve Streptococci -ve: 

Catalase is responsible for the breakdown of hydrogen peroxide into oxygen and water : Staphylococci + ve Streptococci - ve

Bacteriophage typing of Staphylococcus aureus: 

Bacteriophage typing of Staphylococcus aureus Bacteriophage typing of Staphylococcus aureus has provided much valuable information on the epidemiology of hospital-acquired infections. Certain strains of specific bacteriophages , are capable of lysing strains of Staph. aureus . Several distinct bacteriophages have been isolated, propagated in culture, and accepted for typing. They are known by numbers, letters, or a combination of numbers and letters, such as 80, 81, 3B, 3C. The procedure is to coat a culture plate with the "unknown" strain of S aureus , add a drop of each standard bacteriophage to different areas of the plate, and several hours later identify where inhibition has occurred. S aureus bacteriophage type 80/81, for example, is a strain inhibited by bacteriophages 80 and 81. Today there are 27 standard bacteriophages in diagnostic use, and most strains of S aureus can be identified by their susceptibility patterns .

Streptococci spp.: 

Streptococci spp.

Beta- hemolytic streptococci: 

Beta- hemolytic streptococci

Alpha- & gamma hemolysis :: 

Alpha- & gamma hemolysis : Gamma- hemolysis ( no hemolysis ) Many streptococci Alpha- hemolysis : Strep. Pneunomiae & viridans streptocicci

Bacitracin sensitivity : Strep. Pyogenes sensitive : other Streptococci resistant : 

Bacitracin sensitivity : Strep. Pyogenes sensitive : other Streptococci resistant

The Optochin Test: 

The Optochin Test Optochin ( ethylhydrocupreine hydrochloride), a quinine derivative, has a detergent-like action and causes selective lysis of pneumococci . A sterile disk impregnated with optochin is placed on the first sector of an isolation plate before incubation. A zone of inhibition (area with no growth) of 14 mm. or more in diameter will be seen around the disk after incubation if the organism is Streptococcus pneumoniae . Other alpha-hemolytic streptococc i ) Viridans group )are resistant to (not killed by) optochin . Their colonies will thus grow right up to the disk of optochin or have zones of inhibition less than 14 mm. in diameter.

The Bile Solubility Test: 

The Bile Solubility Test Bile will selectively lyse colonies of Streptococcus pneumoniae while other strep are immune to bile's activity. When a bile salt such as desoxycholate is added directly to Streptococcus pneumoniae growing on an agar plate or in a broth culture the bacteria will lyse and the area become clear. Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).

Lancefield grouping: 

Lancefield grouping Streptococcal Grouping Kit, rapid latex agglutination test for Streptococcus Lancefield grouping, includes A,B,C,D,F,G ..etc latex, controls, extraction reagents, cards, and mixing sticks, 60 tests, by Hardy Diagnostics

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NESSERIA

Enteric gram negative bacilli: 

Enteric gram negative bacilli

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On MacConkey Agar : E. coli produce small nonmucoid pink color colonies

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Klebseilla showing pink ( Lactose fermenting ) large size mucoid colony .

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N.L.F. e . g .Salmonella, Shigella and Shigella give yellow colonies

Nutrient agar : Pseudomonas aerogenosa :green coloured colonies due to a greenish pigment pyocyanin produced : 

Nutrient agar : Pseudomonas aerogenosa : green coloured colonies due to a greenish pigment pyocyanin produced

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SWARMING of Proteus spp. forming a thin membranous growth which covers the surface of the medium. These are also Present in waves.

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E. coli on Eosin Methylene Blue ( EMB ). Metallic sheen characteristic color

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Indole test ( Negative and Positive result , Red ring

Methyl red test : red color- positive ( E. Coli ) ; no change - negative ( Klebsiella ): 

Methyl red test : red color - positive ( E. Coli ) ; no change - negative ( Klebsiella )

Voges-Proskauer test : red color – positive ( Klessiella ); no change-negative ( E .coli ): 

Voges-Proskauer test : red color – positive ( Klessiella ); no change-negative ( E .coli )

Citrate utilization test: 

Citrate utilization test Citrate is a source of carbon and is the only source in the synthetic medium known as Simmons Citrate agar. Because this is a synthetic, the source of nitrogen is also controlled and consequently the nitrogen is supplied only by ammonium salts.

Triple Sugar Iron (TSI) : 

Triple Sugar Iron (TSI) Type: Multi-purpose, differential Purpose: Detects glucose, lactose, sucrose fermentation; gas and H 2 S production. Interpretation: Yellow butt, red slant (K/A) = ferments glucose only; yellow butt and slant (A/A) = ferments glucose + lactose and/or sucrose; red but and slant (K/K) = non- fermenter of all 3 sugars; black (+) = H 2 S; bubbles (G) = gas production

TSI agar slant results: (from left) preinoculated (as control),  P. aeruginosa- K/K, E. coli → A/A+,  SalmonellTyphimurium-K/A+G,  Shigella flexneri- K/A : 

TSI agar slant results: (from left) preinoculated (as control), P. aeruginosa - K/K, E. coli → A/A+, SalmonellTyphimurium - K/A+G, Shigella flexneri - K/A

Urease test : red color-positive ( Proteus ) ; yellow color- negative ( Salmonella ): 

Urease test : red color -positive ( Proteus ) ; yellow color - negative ( Salmonella )

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API SYSTEM (Analytic Prophile Index ).

Chorio- allantoic membrane (CAM ): pocks lesions( raised white necrotic foci, caused by poxviruses and herpesviruses: 

Chorio - allantoic membrane (CAM ): pocks lesions( raised white necrotic foci, caused by poxviruses and herpesviruses

Parvoviruses: Single-stranded DNA, nonenveloped viruses : Human parvovirus: Fifth disease ;Anemia in immunocompromised pt.s : 

Parvoviruses : Single-stranded DNA, nonenveloped viruses : Human parvovirus: Fifth disease ;Anemia in immunocompromised pt.s

Papovaviruses : DNA, double-stranded , circular . virion is naked icosahedron Papilloma virus - causes papillomas (warts) by infecting and causing hyperplasia of squamous epithelial cells. BK virus - causes a nephritis and/or urethritis JC virus - progressive multifocal leukoencephalopathy, & colorectal carcinoma : 

Papovaviruses : DNA, double-stranded , circular . virion is naked icosahedron Papilloma virus - causes papillomas (warts) by infecting and causing hyperplasia of squamous epithelial cells. BK virus - causes a nephritis and/or urethritis JC virus - progressive multifocal leukoencephalopathy , & colorectal carcinoma

Adenoviruses :double-strand DNA, naked Respiratory infections in humans , gastroenteritis,Tumors in animals : 

Adenoviruses :double-strand DNA, naked Respiratory infections in humans , gastroenteritis,Tumors in animals

Poxviruses: Smallpox, molluscum contagiosum, cowpox: 

Poxviruses: Smallpox, molluscum contagiosum , cowpox

Herpesviruses : ( Double-stranded DNA, enveloped viruses ) : Simplexvirus (HHV1 and HHV 2) ,Varicellavirus (HHV 3), Lymphocryptovirus (HHV 4),Cytomegalovirus (HHV-5), Roseolovirus (HHV 6), HHV 7, Kaposi's sarcoma (HHV 8) Some herpesviruses can remain latent in host cells: 

Herpesviruses : ( Double-stranded DNA, enveloped viruses ) : Simplexvirus (HHV1 and HHV 2) , Varicellavirus (HHV 3), Lymphocryptovirus (HHV 4), Cytomegalovirus (HHV-5), Roseolovirus (HHV 6), HHV 7, Kaposi's sarcoma (HHV 8) Some herpesviruses can remain latent in host cells

HBV ( Hepadnaviruses ) :- Chronic Persistent Hepatitis asymptomatic - Chronic Active Hepatitis – symptomatic exacerbations of hepatitis - Cirrhosis of Liver - Primary Hepatocellular Carcinoma : 

HBV ( Hepadnaviruses ) :- Chronic Persistent Hepatitis asymptomatic - Chronic Active Hepatitis – symptomatic exacerbations of hepatitis - Cirrhosis of Liver - Primary Hepatocellular Carcinoma

Picornaviruses :Single-stranded RNA, + strand, nonenveloped -Enteroviruses include poliovirus and coxsackievirus -Rhinovirus , Hepatitis A virus : 

Picornaviruses : Single-stranded RNA, + strand, nonenveloped - Enteroviruses include poliovirus and coxsackievirus - Rhinovirus , Hepatitis A virus

Coronaviruses :Single-stranded RNA, + strand, enveloped. Upper respiratory infections &SARS : 

Coronaviruses : Single-stranded RNA, + strand, enveloped. Upper respiratory infections & SARS

Orthomyxoviruses : single RNA strands segmented. Influenzaviruses (Influenza viruses A and B) , Influenza C virus : 

Orthomyxoviruses : single RNA strands segmented. Influenzaviruses (Influenza viruses A and B) , Influenza C virus

Rhabdoviruses :Single-stranded RNA, – strand, enveloped. rabies virus. : 

Rhabdoviruses : Single-stranded RNA, – strand, enveloped. rabies virus .

Paramyxoviruses : Single-stranded RNA, – strand, enveloped, helical. causes parainfluenza, mumps and measles . : 

Paramyxoviruses : Single-stranded RNA, – strand, enveloped, helical . causes parainfluenza , mumps and measles .

HIV :Single-stranded +ve RNA, two identical RNA strands, produce DNA. Lentivirus (HIV) Oncogenic viruses Use reverse transcriptase to produce DNA from viral genome Includes all RNA tumor viruses : 

HIV : Single-stranded + ve RNA, two identical RNA strands, produce DNA. Lentivirus (HIV) Oncogenic viruses Use reverse transcriptase to produce DNA from viral genome Includes all RNA tumor viruses

Arenaviruses : contain helical, segmented single-stranded RNA , enveloped, contain host ribosomes. includes the agent of lymphocytic choriomeningitis & lassa fever virus: 

Arenaviruses : contain helical, segmented single-stranded RNA , enveloped, contain host ribosomes . includes the agent of lymphocytic choriomeningitis & lassa fever virus

Bacteriophage : bacterial viruses, can be RNA or DNA. Used in identification of bacterial types and as a vector in genetic engineering for gene transfer by transduction: 

Bacteriophage : bacterial viruses, can be RNA or DNA. Used in identification of bacterial types and as a vector in genetic engineering for gene transfer by transduction

Yeast colonies: Candida spp.: 

Yeast colonies: Candida spp.

Mold colonies: 

Mold colonies

Aspergillus spp. Colonies on sabouraud agar: 

Aspergillus spp. Colonies on sabouraud agar

Penicillium spp. on sabouraud agar : 

Penicillium spp. on sabouraud agar