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micro lab midterm 1
| Question | Answer |
|---|---|
| E. coli | bacilli/coccobacilli occuring singly |
| S. Epidermis | cocci in pairs (diplococci) and in clusters (staphylococci) |
| R. rubrum | spirilla occuring singly |
| B. Cereus | bacilli occurring singly and in chains (streptobacilli) |
| GRAM STAIN - OBJECTIVE | Classification of bacteria into two major groups based upon chemical and physical nature of the bacterial cell wall. |
| GRAM STAIN - METHOD | Primary Dye = Crystal Violet 2. Add several drops of Mordant –IODINE 3. Decolorize with Ethyl Alcohol [EtOH] 4. Counterstain – SAFRANIN |
| GRAM STAIN - RESULTS | • Gram positive bacteria – Should appear BLUE/PURPLE under oil • Gram negative bacteria – Should appear PINK/RED under oil. |
| ENDOSPORE STAIN | 1. Primary dye – Malachite Green 2. Mordant – steam from boiling water bath add bililous paper 3. Remove slide and cool 4. Wash well with Distilled Water 5. Counterstain – Saffranin |
| ENDOSPORE STAIN - OBJECTIVE | differentiation of bacteria into 2 groups based upon ability or inability to producce a unique, dormant and resistant structure - endospore. |
| ENDOSPORE STAIN - RESULT | Bacillus Subtilis - broth culture = pink chains |
| ENDOSPORE STAIN - RESULT | Bacillus "species" - agar plate culture = green with pink surrounding cell walls |
| SPORE-FORMING BACTERIA | G+ bacilli or streptobacilli (genus bacillus or genus clostridium) |
| NON SPORE FORMING BACTERIA | all others |
| ACID FAST OBJECTIVE | differentiation of bacteria into 2 groups based upon presence or absence of a unique cell wall lipid component |
| ACID FAST METHOD | 1. Primary dye – Carbol Fuchsin 2. Mordant – steam from boiling water bath 3. Remove slide and cool 4. Decolorizer – Acid Alcohol 5. Wash well with Distilled Water 6. Counterstain – Methylene Blue 7. Wash well with Distilled Water |
| ACID FAST RESULT | Mycobacterium Lacticola = rod shaped variation |
| ACID FAST RESULT | Staphylococcus Aureus = clumped round blue spheres |
| ACID FAST BACTERIA | Genus Mycobacterium & Genus Nocardia |
| BACTERIAL MOTILITY DETERMINATION - METHOD | DIRECT: hanging drop slides or wet mount slides |
| BACTERIAL MOTILITY DETERMINATION - METHOD | INDIRECT: media inoculation and incubation |
| BACTERIAL MOTILITY DETERMINATION - METHOD | MOTILITY S MEDIUM - features: semisold 0.75% - agar |
| BACTERIAL MOTILITY DETERMINATION - INDIRECT - REDOX INDICATOR | TTC |
| BACTERIAL MOTILITY DETERMINATION - RESULT | NON MOTILE - Staphylococcus aureus - only center pink/red line |
| BACTERIAL MOTILITY DETERMINATION - RESULT | MOTILE - Pseudomonas aeruginosa, E Coli, Salmonella Typhimurium - pink/red |
| BROWNIAN MOVEMENT | not true motility. Slow, random vibrational movement |
| TRUE MOTILITY | presence of locomotor structure - flagellum. Rapid, directed movement |
| DETERMINATION OF O2 REQUIREMENTS OBJ | classification of bacteria into major groups based upon requirements for O2 for growth |
| STRICT (OBLIGATE) AEROBES | require 20% O2 for growth [AEROBIC RESPIRATION] |
| FACULTATIVE ANAEROBES | growth + O2 [AEROBIC RESPIRATION] |
| FACULTATIVE ANAEROBES | growth - O2 [ANAEROBIC RESPIRATION OR FERMENTATION] |
| STRICT (OBLIGATE) ANAEROBES | growth only - O2. O2 toxic! [FERMENTATION] |
| DETERMINATION OF O2 REQUIREMENTS - METHODS | AGAR PLATE, GAS PAK, TUBE METHOD |
| O2 - AGAR | inoculate your 3 selected organisms in duplicate into labeled sectors of 2 NA plates. 2. Label 1 plate +O2 3. Label other plate -O2 [goes into GAS PAK POUCH] |
| O2 - GAS PAK - REDUCING AGENT | reducing agent - H2 |
| O2 - GAS PAK - REDOX INDICATOR | redox indicator - Methylene Blue [oxidized = blue, reduced = colorless] |
| O2 - TUBE METHOD | carefully loop stab to incolate all 3 selected organisms into separate tubes of FTB. Screw tops lightly. |
| O2 - TUBE METHOD - MEDIUM | MEDIUM - Fluid Thioglycollate Broth (FTB) |
| O2 - TUBE METHOD - REDUCING AGENT | reducing agent - Thioglycollate (-SH) |
| O2 - TUBE METHOD - REDOX INDICATOR | redox indicator - Resazurin [oxidized = PINK, reduced = colorless] |
| O2 RESULT | Strict anaerobe [clear on top, red on bottom] |
| O2 RESULT | Facultative anaerobe [both top and bottom are red] |
| O2 RESULT | Strict Aerobe [red on top, clear on bottom] |
| CULTURAL CHARACTERISTICS - MEDIA | Nutrient Agar Slant, Nutrient Broth, Nutrient Agar Plate, Nutrient Gelatin Tube |
| CULTURAL CHARACTERISTICS - NUTRIENT AGAR PLATE | colonial characteristics configuration, margins, elevations |
| CULTURAL CHARACTERISTICS - NUTRIENT AGAR PLATE - METHOD | Streak to isolate known and enteric unknown (fr Fresh suspension) on separate NA plate by using the Quadrant method. |
| CULTURAL CHARACTERISTICS - NUTRIENT AGAR PLATE - RESULT | under Microscope, observe for size, color, opacity, form, elevation and margin. |
| CULTURAL CHARACTERISTICS - GELATIN TUBE - REACTION | Gelatin (H. Mol Wt Protein) with gelatinase ==> (gelatinase) Amino Acids soluble |
| CULTURAL CHARACTERISTICS - GELATIN TUBE - METHOD | heavily loop inoculate known and unknown into separate tubes of nutrient gelatin. |
| CULTURAL CHARACTERISTICS - GELATIN TUBE - RESULT | recover nutrient gelatin tubes and refrigerate for 20+ minutes. 2. Immediately observe: a) if medium solidified ==> gelatin present ==> gelatinase - b) if medium liquified ==> gelatin absent ==> gelatinase + |
| BIOCHEMICAL CHARACTERISTICS - FERMENTATION | 1. incomplete oxidation of simple sugars, 2. production of detectable by-products (acidic, alkaline, +/- gas) |
| BC- FERMENTATION MEDIUM | Phenol Red Carbohydrate Broth |
| BC- FERMENTATION FEATURE | sole fermentable C+ Energy source |
| BC- FERMENTATION PH INDICATOR | PHENOL RED |
| BC- FERMENTATION GAS INDICATOR | DURHAM TUBE |
| BC- FERMENTATION MEDIUMS | Phenol Red Glucose broth , Phenol Red Lactose broth, Phenol Red Sucrose broth, MR-VP broth |
| BC- FERMENTATION MR-VP | no pH indicator - glucose is present |
| BC- FERMENTATION MR-VP - METHYL RED | glucose ==> Mixed Acid Fermentation (Aceitic, Formic, Sucenic ACID) + Methyl Red Test |
| BC- FERMENTATION MR-VP - VP | glucose ==> Alcoholic Fermentation (2,3 butanediol) + Voges Proskain test |
| BC- FERMENTATION MR-VP - RESULT- METHOD | inoculate known and unknown to tubes. With the use of pipette, take 1ml out of tube into non sterile tube |
| BC- FERMENTATION MR-VP - RESULT- METHOD - MR | in the original tube, add 8-10 drops of METHYL RED a) If yellow color ==> pink or red (ACIDS ARE INDICATED, +MR) b) If yellow color ==> yellow (NO ACIDS, -MR) |
| BC- FERMENTATION MR-VP - RESULT- METHOD | in non sterile tube, add 18 drops each of Barritts A & B solution, mix well to aerate a)If yellow ==> pink, red (ALCOHOL INDICATED, +VP) b)if yellow ==> yellow (NO ALCOHOL INDICATED, -VP) |
| BC- FERMENTATION - PHENOL RED - RESULT | GROWTH , no pH change (pink, purple) ==> - |
| BC- FERMENTATION - PHENOL RED - RESULT | GROWTH, pH change (pink - bright yellow) ==> +A or + on chart |
| BC- FERMENTATION - PHENOL RED - RESULT | GROWTH, pH change + gas in Durham Tube ==> +A/G |
| BC- SPECIFIC ENZYME - CATALASE RX | 2 hydrogen peroxide with catalase produces water and oxygen |
| BC- SPECIFIC ENZYME - CYTOCHROME C OXIDASE RX | cytochrome reduced with oxidation produces cyto c oxidized |
| BC- SPECIFIC ENZYME - METHOD | loop streak to inoculate known and unknown unto separate sodes of TSA (TRYPTICASE SOY AGAR) plate. Just one streak! |
| BC- SPECIFIC ENZYME - RESULT - CATALASE | recover TSA plates 2) add 1-2 drops of 3% H₂O₂ to growth plate 3) if fizz, bubble + O₂ ==> catalase + 4) if - O₂ ==> catalase - [ie enterococcus faccalis] |
| BC- SPECIFIC ENZYME - RESULT - CYTOCHROM C OXIDASE | utilize sterile toothpick to sample growth from plate and transfer to a window of DRY SLIDE 2) if blue/purple with in 20 secs ==> oxidase + [pseudomonas aeruginosa] 3) if blue/purple after 20 secs ==> oxidase - |
| BC - NITRATE REDUCTASE - RX | Nitrate (NO₃) with nitrate reductase produces Nitrite (NO₂) and water |
| BC - NITRATE REDUCTASE - METHOD | loop inoculate known and unknown onto separate tubes of nitrate broth |
| EXOENZYMES | synthesized intracellularly, utilized extracellularly |
| EXOENZYMES - CARBOHYDRATES - GENERALIZED RX | polysaccharide ==> monosaccharides and dissacharides |
| EXOENZYMES - CARBOHYDRATES - SPECIFIC ENZYME | Amylase |
| EXOENZYMES - CARBOHYDRATES - Reaction | starch ==> glucose + maltose |
| EXOENZYMES - CARBOHYDRATES - Medium | starch agar plate (SA) |
| EXOENZYMES - CARBOHYDRATES - Method | loop streak inoculate known and unknown onto separate sides of SA plate. |
| EXOENZYMES - CARBOHYDRATES - RESULT | ADD LUGOL'S IODINE b) dark blue (surrounding) c)Halo = yes = Amylase +d) Halo = no = Amylase - |
| EXOENZYMES - PROTEASES - GENERALIZED RX | protein ==> 20 Amino Acids |
| EXOENZYMES - PROTEASES - SPECIFIC ENZYME | Caseinase (milk protein) |
| EXOENZYMES - PROTEASES - Reaction | Casein ==> 20 Amino Acid |
| EXOENZYMES - PROTEASES - Medium | Skim Milk Agar Plate (SM) |
| EXOENZYMES - PROTEASES - RESULT | Halo = yes = Caseinase + b) Halo = no = Caseinase - |
| EXOENZYMES - SPEC ENZYME - LIPASES - RX | Complex lipid ==> triglycerides ==> glycerol + fatty acids |
| EXOENZYMES - SPEC ENZYME - NUCLEASES - RX | DNA =DNAase==> Deoxyribonucleotides; RNA =RNAase==> Ribonucleotides |
| ENDOENZYMES | synthesized and utilized intracellularly |
| ENDOENZYMES - ROLES | catalyze the 1000 of specific biochemical reaction of cellular metabolism |
| ENDOENZYMES - TRYPTOPHANASE (TSA) - reaction | Tryptophan ==> Indole + pyrovic acid + Ammonia |
| ENDOENZYMES - TRYPTOPHANASE (TSA) - MEDIUM | 1% tryptonic broth |
| ENDOENZYMES - TRYPTOPHANASE (TSA) - Method | loop inoculate known and unknown unto separate tubes |
| ENDOENZYMES - TRYPTOPHANASE (TSA) - RESULT | ADD KOVACZ REAGENT a) growth = yes = + b)growth = no = - |
| ENDOENZYMES - UREASE - reaction | Urea ==> Ammonia + CO₂ |
| ENDOENZYMES - UREASE - MEDIUM | Urease broth |
| ENDOENZYMES - UREASE - pH indicator | Phenol Red - if urease is produced, it will become alkaline. |
| ENDOENZYMES - UREASE - Method | heavily loop inoculate known and unknown into separate tubes |
| ENDOENZYMES - UREASE - RESULT | pink/purple = Urease + b) yellow = Urease - |
| ENDOENZYMES - CYSTEINE DESULFURASE - reaction | Cysteine ==> H₂S, pyrovic acid and ammonia |
| ENDOENZYMES - CYSTEINE DESULFURASE - MEDIUM | Kliglers Iron, Agar Slant KIA |
| ENDOENZYMES - CYSTEINE DESULFURASE - Method | needle stab to inoculate known and unknown into separate tubes |
| ENDOENZYMES - CYSTEINE DESULFURASE - RESULT | black precipitate = H₂S+ a) all yellow = Lactose +, glucose - b)red/yellow = lactose +, glucose + |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - reaction | Citrate ==> Growth + Alkaline rx due to ammonia |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - MEDIUM | Simmons Citrate Agar slant |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - RX | Citrate ==> Sole C & Energy Source |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - RX | Ammonia Salts ==> Sole N Source |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - pH indicator | Brom Thymol blue |
| ENDOENZYMES - CITRATE UTILIZING ENZYME - Method | loop streak known and unknown unto separate slants. |
| BC - NITRATE REDUCTASE - RESULT | recover nitrate broths and add 5 drops each of RGT A, RGT B and mix gently a) if yellow ==> pink red ==> Nitrite present ==> Nitrate reductase + |
| BC - NITRATE REDUCTASE - RESULT 2 | b)if PALE yellow ==> yellow persist ==> Nitrite absent [ADD RGT C - ZINC DUST] c)If yellow ==> pink/red ==> nitrate reductase - d) if yellow ==> yellow ==> Nitrate reductase + |
Created by:
jekjes
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