Micro Laboratory Midterm Review

Micro Laboratory Midterm Review Case Studies 1. 24 y/o Female MechanicDeep wound on L palm, oozing pus. Purple/red streaks running up the L arm. Febrile 3 days later, V/D, enlarged nodes. *Staphylococcus aureus V/D confirms S. aureus  TSS Other Info/ChoicesStreptococcus pyogenes & Streptococcus aureus both live on the skin and can easily enter a wound when skin is broken causing problems like cellulites etc. Clostridium causes gas gangrene and tetni. Pasteurella multocida is acquired via animal bites and causes severe upper respiratory infections. The other choice was Pseudomonas. 2. 27 y/o Female Ecology Student August, fever, fatigue, tender joints, headache, stiff neck, backache, circular “Bulls eye” rash (erythema migrans) only 75% of cases, and an arrhythmia ( late s/s of disease). PT Hx-Wisconsin May-June, camping, Ticks, and two weeks until onset of Sx. Borrelia burgdorferi (Lyme Disease) STAGE II Antibiotic Tx: Ampicillin, Ceftriaxone, Erythromycin, Amoxicillin, Doxycycline, or Z Pack. Other info/choices Stages of disease I. Flu like Sx II. Cardiac/Nerve Sx. III. Nerve Damage/Meningitis Sp/AgTesting Antibody Primary or Secondary defense “Antibodies may be there from previous infection” Antigen testing is 100% because if present organism must be there! Vaccine for Lyme disease is no longer given, because of cost. NOT from Deer ticks, from White Footed MouseTickHuman. 3. 5 y/o WDM Child Fever, sore throat x 24h, Swelling and drainage of the conjunctiva, tonsils w/ white patchy excaudate, swollen AC nodes. Streptococcus pyogenes GAS Group A Strep, ß-hemolytic. “Tonsillopharyngitis”. Tx with Antibiotics: Penicillin or Erythromycin x 10 days. Prevents Scarlet/Rheumatic feverCardiac and kidney problems. Rapid Strep should be followed by C&S. 4. 22 m/o Lethargic Female Running nose, hoarse cough, and fever x 48h. Cloudy eyes and otitis media (Not Significant) Throat red and covered c mucus. Larynx swollen and raw. Rapid Strep Negative. Adenoviral pharyngitis (Croup) S/S point to viral, Neg Strep test, and good response to vapors in room. 80% or higher of Croup is caused by virus rather than bacteria.

Tx-Vaporub, analgesics, and decongestants. Antibiotics should not be given unless C&S are Positive for Strep or Hib. Other Possible Dx Mono ( little or NO Sx in Young children because of immature immune system) RSV, Rhinovirus, Parainfluenza 1 & 3, Coxsackie’s Virus. 5. 22 y/o Male College Student C/C Chest Pn & Fever. Pale, tired, EKG normal, Chest X ray is Neg for consolidation, tight productive cough, muscle aches, headache, and fever x 36 hours. Rapid strep Neg. Tonsils and adenoids have been removed. Influenza Can  bacterial pneumonia. Tx- Antipyretics, Rest, hydration, and Tamaflu c in two days of Sx onset. Prophylactic Vaccine Influenza A (2) B (1) & C (not common) Other info: Malaria is caused by a protozoan. Asia and India has high incidences of flu because of the population and living c in close proximity of birds, pigs, & cows. Research is suggesting that the 1918 Flu pandemic (150 Million deaths) originated c in birds. 6. 68 y/o Male c 1˚ Dx of Alzheimer’s Lethargy, shallow paradoxal breathing, fever (102.4), Resp. rate of 33 (N12-20), LLL Consolidation, Chills, and Acid Fast (TB) Negative. Green bloody sputum. Klebsiella pneumoneae “Old man’s friend” Aspiration bacterial pneumonia. Gram Negative Encapsulated Rods. *Cytotoxic damage to lungs is a classic Sx. Tx- 3rd generation cephalosporins and aminoglycosides Other info: Klebsiella is Normal c in intestinal floraRarely causes Pneumonia, more often UTI. Tuberculosis is caused by Mycobacterium tuberculosis. 7. 35 y/o Male Accountant Burning R epigastric PN1-3 hours post meals. Vomiting of frank blood, afebrile, no wt loss, slight rebound tenderness, and Positive occult blood test. Helicobacter pylori Gastric Ulcer  MALT Lymphoma(Malignant growth on tissue c in stomach and intestines, caused by prolonged infection) Tx- H+ Pump Inhibitors, Bismuth citrate, and amoxicillin and clarythromycin. Stage ulcer (Endoscopy), and monitor bleeding. *Possible Surgery? Other Info: Salmonella  GE and Crohn’s Mycobacterium paratuberculosis (Anemia). Breath test “Urea”

8. 4 y/o Female Bloody diarrhea, fever 39C, and vomiting x 24h. No urination for 12 hours. 4 days prior to Sx, child went on daycare field trip, ate ground beef, fries and soda. Dehydrated, poor kidney function (bld test), and lysed RBC’s in urine(UA). Escherichia coli (0157:H7) EHEC O & H are antigen markers. Bacterial Enteritis. Pathogenesity is caused by Cytotoxins r/t Shigella dysenteriae type I or type II., Cholera et al. (This happens because a virus will lysogenize E. coli c other bacteria that make cytotoxins) Tx- Antidiarrheals and NO antibiotics because of kidney function. Other info: EHEC HUS (Hemolytic Uremic Syndrome), GE (Gastro Enteritis), Dysentery (Shigella), and HC (Hemorrhagic Colitis). 9. 26 y/o Female Genital itching and sharp severe pain on the labia, c 3 similar episodes over the past 3 months. Following each episode sores appeared and crusted s leaving scars. No vaginal discharge. Urine clear and yellow c No abnormalities/ bacteria. Temp 36.5 C. Pt Hx- Unmarried, sexually active, Oral contraceptive x 4 yrs. 5 partners c in past year, c episodes becoming progressively worse. HSV I or II Genital Herpes Can be transmitted up to 2 wks prior to outbreak, Use condoms always, No sex in presence of lesions/zoster. Tx- None….Preventative antiviral medication which inhibits viral DNA synthesis and makes time between outbreaks shorter and the outbreak itself shorter. Other info: HSV I & II are only 50% related. Genital herpes can be caused by either, and spread from mouth to genitals and visa versa. Out breaks in mouth “Cold sores” come from HSV lying dormant c in the trigeminal nerve (CN V). Genital Sores come from HSV that lays dormant c in the sacral ganglia. Having HSV increases a women’s risk for cervical CA, and increased risk for men and women to get Herpes Encephalitis. Benson’s Lab Manual CHPT 1 Brightfield Microscopy Brightfield microscopeAllows light rays to pass directly through the eye s being deflected by a plate c in the condenser. *Voltage controlIntensity of the light *Neutral density filterReduce light intensity below the lower limits of the voltage control. *Lens system *OccularEye piece c 2 or more internal lenses(10x) *ObjectivesScanning(4x)Low Power(10x)High Dry(40x) and Oil immersion(100x) *CondenserDirects light from lamp to slide

*Diaphragm(c in condenser) Regulates amount of light that can get to the slide. When Diaphragm is closed the contrast and resolution increase, while the brightness is decreased. *Focusing1st focus c course adjustment, 2nd c fine adjustment *Diopter Adjustments(distance between pupils)1st focus c R eye, 2nd focus L eye by turning the diopter adjustment ring on the eyepiece until a sharp image is seen. *Resolution or resolving powerTwo objects will be seen as separate entities (the optimum resolution of the best microscopes c oil immersion is 0.2µm). *Blue (Green) FilterCan be placed over the light source to improve resolution because, of the colors short wavelengths. *The condenser should be kept at its highest position(allowing the most light) in order to improve resolution. *When the diaphragm is stopped down to much it improves contrast, yet reduces numerical aperture. ***If magnification is increased, then the resolution must be increased. Increasing to a 20x ocular, WON’T increase resolution. *Solvents for cleaningGreen Soap and H2O (preferred). Xylene(Not used in our lab… Toxic). Acetone and alcohol(may damage glue/cement). *Bringing your specimen in to focusStart c minimum voltage and magnification(objective), center specimen in light source, 1st focus c coarse adjustment(lowest objective only), 2nd fine adjustment, then increase objectives until specimen is at desired magnification. *Parfocal ScopesImage will remain in focus when changing to higher powered objectives. *When increasing lightOpen diaphragm 1st, then increase voltage if needed. This increases life time of lamp bulb. *Oil Immersion has the same refractive index as glass, therefore saving light rays from defraction. *Never take apart any part of the scope for cleaning. *When moving to a higher objective such as oil immersion, there is NO need to increase distance between the slide and the lens. *Increased Magnification = Shorter Working Distance CHPT 2 Darkfield Microscopy The darkfield microscopeIdeal for viewing transparent living organisms i.e. Syphilis spirochetes. Also used c fluorescence. *Achieved by blocking the light below the condenser with a star diaphragm or cardiod condenser. This allows only oblique rays to strike the specimen. The background will be completely black. *Star diaphragmFits into the filter slot of the condenser housing. Can be made with celluloid/glass disks and opaque paper(colored paper for colored background). Good for LG organisms c low objectives. Keep diaphragm wide open and voltage on high. *Cardioid CondenserLight rays entering the condenser are 1st reflected off of a convex mirror surface and 2nd a concave surface to get desired oblique rays. (Provides greater light concentration, and is good for high-dry and oil immersion objectives.

CHPT 3 Phase-Contrast Microscopy Phase-Contrast MicroscopeFrederick Zernike1933, originally the “Zernike microscope”. Differentiates the transparent protoplasmic structures and enhance the contrast between the cell and it’s surroundings. WITH OUT HAVING TO STAIN THE ORGANISM. *Microscope of choice for viewing living cells on a slide with a cover glass and motility. *Objects will be categorized as Phase or Amplitude. *Amplitude objects show up dark because the intensity of light rays (amplitude) is reduced as light passes through objects, i.e. brightfield microscope. *Phase objects are completely transparent since light rays pass through them unchanged with respect to amplitude. A ¼ phase shift occurs s any change in amplitude. Direct RaysPass through medium unaffected. Diffracted RaysBend as they pass through a medium due to its density. **Direct and diffracted rays can be brought in to exact phase or coincidence, as result a converged ray is the sum of the two waves or Amplitude summation. If two rays of equal amplitude are in Reverse phase ½ of a wavelength off, then they cancel each other out, causing a dark object, AKA Interference. *A Zernike Scope has a different type of diaphragm and a phase plate in comparison c the brightfield scope. *The diaphragm consists of an annular stop allowing a hollow cone of light that passes through the condenser to the medium. *The phase plate contains a phase ring that advances or retards the direct light rays by ¼ wavelength. *In bright phase and dark phase microscopy, the images light is proportional to the square of the amplitude. Thus they will appear 4x brighter or darker than in the regular microscope. *The ocular of the Phase-Contrast scope can be replaced with a centering telescope in order to view the orientation of the phase and annular rings. *Stained/Prepared slides will not be ideally viewed. *Brining specimens in to focus (wet mount)focus using brightfield at low magnification, then switch to phase at the same magnification, Adjust light c diaphragm, then voltage regulator. *The “Halo Effect” is normal with phase objects. *Direct rays are altered by the phase ring on the phase plate. * + ¼ or – ¼ phase shift will occur when light rays emerge from a transparent object. *If direct rays passing through an object are advanced by ¼ wavelength by the phase ring, then the diffracted rays are ½ wavelength OUT of phase with c the direct rays and in reverse phase. CHPT 8 Aseptic Technique *The use of aseptic technique insures that no contamination of organisms c in culture materials, or those handling contagious organisms. Aseptic Technique 1. Disinfect work area c bleach water, will kill vegetative cells and viruses, but may not kill endospores.

2. Sterilize inoculation loops c Bunsen burner(open flame) or incinerator, until tip of metal becomes RED hot (about 30 seconds), this will kill any organism that may be present. 3. *Culture Tube InoculationFlame the mouth of culture tubes prior to inserting loops. If broth tube insert loop and twist several times. If it has an agar slant, drag loop from bottom to top of slant. If it is an agar stab, stab the needle in to the medium. 4. Before recapping make sure to reflame mouth of tube. 5. *Petri PlateLoops are used to inoculate plates. Hold the lid diagonally over plate to protect surface from contaminates in the air. The loop should be GENTLY dragged over the agar surface, do NOT gouge or disturb surface. Cover Plate. Sterilize the loop. 6. Finally Disenfect your work area with bleach water. *Broth Tube Broth tube inoculation (Escherichia coli) 1. Disinfect work area c a sponge 2. Label tube of Nutrient broth c E. coli & initials 3. Sterilize loop 4. Skake tube to disperse culture 5. Grasp cap of tube c little finger and flame mouth to sterilize 6. Insert loop 7. Remove loop, reflame, recap, & replace tube in to rack. 8. Grasp sterile nutrient tube c little finger, remove cap and flame mouth 9. Insert loop in to broth, moving it back and forth to disperse E. coli 10. Remove loop, flame mouth, and recap 11. Sterilize loopReturn to container (NOT Desk Top) 12. Incubate culture at 37˚C for 24-48 hours Agar SlantAgar Slant inoculation (Escherichia coli) 1. Disinfect work area c a sponge 2. Label tube of Nutrient agar slant c E. coli & initials 3. Sterilize loop 4. Grasp cap of tube c little finger and flame mouth to sterilize 5. Insert loop 6. Remove loop, reflame, recap, & replace tube in to rack. 7. Flame mouth of sterile agar slant 8. Using a serpentine motion insert loop in to sterile agar slant and gently inoculate the surface. 9. Remove loop, flame mouth, and recap 10. Sterilize loopReturn to container (NOT Desk Top) 11. Incubate inoculated slant at 30˚C for 24-48 hours Petri PlateAgar Slant (Escherichia coli) *Plates are never flamed, when labeling write on bottom of plate. Incubate upside down to prevent condensation on agar surface and spreading of inoculated organism. 1. Disinfect work area c a sponge 2. Label tube of Nutrient agar slant c E. coli & initials 3. Sterilize loop 4. Raise lid of plate diagonally to access culture, DO NOT gouge in to agar with loop as you pick up organisms, close lid. 5. Grasp cap of tube c little finger and flame mouth to sterilize

6. Using a serpentine motion insert loop in to sterile agar slant and gently inoculate the surface 7. Remove loop, flame mouth, and recap 8. Sterilize loopReturn to container (NOT Desk Top) 9. Incubate inoculated slant at 37˚C for 24-48 hours CHPT 10 Smear Prep *Prior to all staining procedures a smear must be done. *When preparing a smear, we want to cause cells to adhere to the slide so that they will not be washed away during staining procedures, avoiding shrinkage of cells (artifacts), and lastly to make the sample thin so that the cells arrangement and details my be viewed. Liquid MediumDrop 1-2 loopfuls of organism directly on slide. Petri Medium1st put 1-2 loopfuls of Distilled H2O on to slide, sterilize loop, then a VERY SMALL Amount of organism is diluted (H2O on slide) and dispersed on slide. *Allows slides to completely air dry *Pass over flame 3x to heat fix and kill organism CHPT 11 Simple Staining (Corynebacterium diptheriae)Book, NOT done in Lab! *Simple staining is best for viewing morphology and the presence or absence of granules. Simple Stainingthe application of a single stain to color an organism, the most common dyes used are methylene blue, basic fuchsin, and crystal violet (ALL BASIC). They all work because the have color bearing ions (chromophores), that are positively charged (cationic). Bacteria have a slightly (-) charge, causing an attraction between dye and organism. *Methylene blue (Methylene+, Chloride-) was used in this lab. 1. Smear prep slide 2. Stain c MB (30 sec-1 min) 3. Carefully flush c distilled H2O 4. Blot dry 5. View c microscope *Acidic dies like Eosin (Sodium+, Eosinate-) contain anionic chromophores will NOT stain organisms because of an electrostatic repelling force. C. diptheriae PleomorphismIrregularity of form, usually rod shaped, it can also be clubshaped, spermlike, or needle-shaped. Metachromatic Granuleswhen stained c MB, reddish-purple granule will show c in cell, masses of volutin, a polymetaphosphate. Palisade arrangementParallel arrangement of rod shaped cells “picket fence”, common to corynebacteria. CHPT 12 Negative Stain *Negative stains with Nigrosin or India ink (Both Basic) are used for morphological viewing of organisms, particularly for Size and presence of a capsule(because there is NO

HEAT FIXATION) Also Good For viewing Spirochaetes i.e. Treponema pallidum and Borrelia burgdorferi ** We viewed molds in this lab *Cells will appear translucent with a dark background. There are 3 methods for this staining 1. Feathering c a spreader slide (drop of ink) 2. Organisms spread over small area of slide c inoculation needle (loop of ink) 3. Woeste-Demchick’s MethodSingle coat of felt tip pen ink over a smear. Proceedure 1. Disinfect area 2. Clean slides (Bon Ami) 3. Choose a method and prepare slide 4. ORALRemove organisms from between teeth c sterile toothpick, break up clusters on slide. 5. CulturesUse sterile loop 6. Spread according to method 7. Air Dry (DO NOT HEAT FIX) CHPT 13 Capsular Stain *A capsule or glycocalyx is a extracellular slime layer the serves as protection for certain bacteria, by preventing phagocytic WBC’s (neutrophils and monocytesmacrophages) from engulfing them. The capsule is a means for attachment for some bacteria, i.e. Streptococcus mutans, which becomes dental plaque. *When staining a capsule combine methods of simple and negative staining. 1. Negative Stain organism 2. Heat fix 3. Apply CV for 1 min. 4. CAREFULLY flush c distilled H2O 5. Blot 6. View c scope Method II 1. Smear 2 loops of organism c CV 2. Air Dry 3. Decolorize c Copper Sulfate (Blue) 4. Blot and view c scope *Capsule will appear as a halo around a purple dot, on a blue background. Capsulated Organisms Haemophilus influenzae Klebsiella pneumoniae Streptococcus bovis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarius Streptococcus viridans

CHPT 14 Gram Stain *In 1884 a Danish physician Christian Gram was trying to develop a staining process that would differentiate eukaryotic cell nuclei from bacterial cells with in tissue samples. Although his tissue stain was not completely successful, he was able to separate bacteria in to two categories, Gram + and Gram -. *Gram + bacteria retain a crystal violet iodine complex through decolorization with ETOH. (Purple) *Gram –bacteria lose their crystal violet iodine complex, when decolorized by ETOH. They must be counterstained with safranin in order to be viewed. (Red) *Primary stainCV *Mordant (forms an insoluble complex in Gram + cells)Iodine *Decolorization c ETOH or Acetone *Gram + cells have a thick peptidoglycan layer c in the cell wall. *Gram – cells have an outer membrane (LPS) that covers a much thinner layer of peptidoglycan. (Determining weather the dye/mordant complex will remain c Gram + organisms, or be removed from the Gram – organisms). *IMPORTANT Things to keep in mind while doing a gram stain *Cultures should be 16-18 hours old, Gram + cultures that are older than this can be converted to Gram -, or Gram variable, giving erroneous results. Gram – can also convert to Gram +. *Smears should be thin, thick smears can affect decolorization by trapping primary stain making Gram – cells appear Gram +. *Make sure not to over decolorize, this can make Gram+ cells appear Gram -. 1. Smear prep slide 2. 1˚stainCV for 1 min. 3. Carefully flush slide c distilled H2O 4. MordantGram’s Iodine for 1 min. 5. Decolorize carefully c ETOH 20-30 seconds 6. Flush carefully c distilled H2O 7. 2˚ CounterstainSafranin for 1 min. 8. Blot and air dry 9. View under scope. *Acid-fast Bacteria i.e. mycobacteria will appear Gram + CHPT 15 Spore Staining *Bacterial species that belong to Genus Bacillus or Clostridium, and exhaust their essential nutrients, they will undergo a resting part of their lifecycle known as endospore form. *Endospores are dehydrated structures that do NOT actively undergo metabolism. *They are resistant to heat, radiation, acids and many chemicals (disinfectant) that would normally kill cells in a vegetative state. *Thick PRO coat called an exosporium *The higher the water content of a spore, the less heat resistant it will be. During sporulation, the H2O content of a spore is 10-30% that of it’s vegetative

state. This occurs because CA+, a spore specific PRO, and dipicolinic acid form to make a gel that controls the movement of H2O in and out of the endospore. *Since endospores are not easily destroyed by heat, they define the conditions for sterility. They must be autoclaved under high pressure (15psi) steam at 121˚C, for 15-20 minutes. *The resistant properties of endospores make them difficult to stain. If stained c CV, the spores will appear as unstained areas c in the vegetative cells. If heat is applied while staining with Malachite Green, the stain penetrates the spore becoming trapped inside. In this case Heat acts as the Mordant (facilitating the uptake of stain.) Schaeffer-Fulton Method (Bacillus megaterium) We used this method in lab (B. cereus and B. subtilis) Spores will appear green c in a pink sporangium 1. Smear prep slide 2. Flush slide c Malachite Green 3. Hot plate for 5 minutes (paper towel optional) DO NOT ALLOW STAIN TO DRY OUT 4. After slide has cooled, flush c distilled H2O for 30 seconds 5. CounterstainSafranin (20 Sec.) 6. Flush 7. Blot dry and view with scope The Dorner spore stain method 1. Make a heavy suspension of bacteria (several loopfuls in 5 drops of distilled H2O) 2. Add 5 drops of carbofucshin to suspension 3. Heat suspension in beaker for 10 min. 4. Mix several loopfuls of suspension c nigrosine on slide 5. Feather c spreader slide 6. Air dry and view with scope CHPT 18 Culture Media Preparation *Cultivation of microorganisms on an artificial growth media require ALL the nutrition and energy needed to sustain life. *Complex MediumExact composition i.e. AA, Vit, and growth factors are UNKNOWN –Nutrient Agar *Defined MediumI.e the one used to grow E. coli, has a Specific KNOWN chemical composition. *All media complex or defined must contain *CarbonHeterotrophs (Meat & Plant extracts. Autotrophs (Fix CO2) *EnergyChemoorganotrophs (Most Bacteria) Breakdown organic molecules via fermentation or respiration *ChemolithotrophsOxidize inorganic ions i.e. nitrate or fe. *Photoautotrophs Convert solar E into organic E. No E is supplied via agar, only c illumination. (cyanobacteria and green/purple Sulfur bacteria.) Photoheterothrophs(Few Bacteria) Derive E from photosynthesis, but derive their Carbon from organic molecules i.e. succinate & glutamate. (purple NonSulfur bacteria)

*NitrogenWhen bacteria can not synthesize their own N, beef extract and peptones can be added to the media. *Rhizobium and Azotobacter can fix atmospheric N. *Minerals/Electrolytes Essential because they are co-factors for enzymes *Most bacteria require catalytic or Sml. Amounts *Vitamins and Growth factors Niacin is a coenzyme for NAD and flavin for FAD. *Strep and lactobacilli are unable to synthesize Vit. *E coli can synthesize it’s own vitamins. *Some bacteria are more fastidious if grown in BLD. or serum (growth factors) Water&0-80% of a cell consists of water. Cells require an aqueous environment for enzymatic reactions to occur. *ALWAYS use distilled H2O when preparing a media, tap water contains electrolytes that will react c peptones and meat extracts. *Most bacteria grow at a neutral pH of 7. Fungi prefer pH values of 5. *Most commercial media do NOT need pH adjustment. *The pH of defined synthetic media can be adjusted c HCL(acid) or Sodium Hydroxide(base). Selective MediaAllows certain bacteria to grow, yet inhibits growth of others. Antibiotics, dyes, and chem. Compounds are added… i.e. *EMB allows Gram – to grow, yet inhibits Gram + bacteria *MSA contains NaCl that selects for S. aureus, while other organisms can NOT tolerate the high salt concentration. Differential MediaContain substances that cause certain bacteria to take on an appearance that distinguishes them from others. *MSAS. aureus ferments mannitol, pH from red to yellow. *EMBGram – bacteria that ferment lactose will turn media metallic green. Agar1st introduced in Koch’s laboratory in 1930, polysaccharides isolated from seaweed is added at 1.5% to solidify liquid media. Melts at 100˚C, yet does not solidify until 45˚C(cells will NOT die at this temp) *Semisoft media0.4% Agar *It is only necessary to weigh out and measure a specific medium i.e. nutrient Agar & dissolve in water(Poss. Adjust pH) *Prior to using media, it must be sterilized, most can be autoclaved. If media contains a heat sensitive component, it can be filtered at 0.45 microns. Gram + Bacillus “Rods” Bacillus anthracis Bacillus cereus Bacillus subtilis Clostridium botulism Clostridium perfringes Clostridium tetni

Gram + Cocci “Spheres” Enterococcus faecalis Kocuria rhizophila(previously Micrococcus luteus)(Tetrococci) Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Streptococcus bovis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarius Streptococcus viridans

Gram – Bacilli “Rods” Citrobacter freuendii Enterobacter aerogenes Escherichia coli Haemophilus aphrophilus Haemophilus influenzae Haemophilus parainfluenzae Klebsiella oxytoca Klebsiella pneumoniae Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas cepacia Salmonella enteritidis Salmonella typhimurium

Shigella flexneri Vibro fischeri Gram -Cocci “Spheres” Nesseria gonorrhoeae Haemophilus aphrophilus Haemophilus influenzae Haemophilus parainfluenzae Gram - Spirochaetes “Spirilium” Treponema pallidum Borrelia burgdorferi