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Clinical Laboratory Science: Microbiology

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Gram Stain
* Stain to detect bacteria & yeast
1. CRYSTAL VIOLET - 1min
2. GRAM'S IODINE - 1min
3. ACETONE-ALCOHOL - 10sec decolorizer
4. SAFRANIN
Polymorphonuclear Neutrophil (PMN)
* Segmented nucleus
* Attracts infected site in response to bacterial & host inflammatory products
* Short-lived
* Phagocytic
* Appears in acute stages of bacterial infection
Lymphocytes
* Smaller than PMNs
* Round nucleus with thin border of cytoplasm
* Common on gram stained CSF
* Large amount in CSF indicates Viral Meningitis
Macrophages
* Slightly larger than PMNs
* Large, slightly indented, non-segmented nucleus
* Polymorphic
* Resembles large, atypical lymphocytes
* Widely distributed in tissues throughout the body
* Long-lived
* Phagocytic
* Prevalent in chronic infections
Squamous Epithelial Cells
* Direct Gram Stains
* Large size
* Small nucleus
* Abundant cytoplasm
Ciliated Columnar Epithelial Cells
* Gram Stains from the LOWER RESPIRATORY TRACT
* CILIA at one end of an elongated shaped cell
Spermatozoa: Sperm cells
* Gram stain from genital or urine specimens
Bacteria:
Gram-Positive Cocci
* SPHERICAL bacterium
Groupings:
* DIPLOCOCCI - pairs (may be elongated)
* CHAINS - varies in length
* TETRADS (small clusters)
* CLUSTERS - groups of clusters
Bacteria:
Gram-Positive Rods/Bacillus
* RECTANGULAR shaped bacterium
Groupings:
* LONG & WIDE with BLUNT ends
* LONG & NARROW with BLUNT ends
* COCCOBACILLI - very short coccoid rods
* BRANCHED - long & narrow w/BEADED staining pattern
Bacteria:
Gram-Negative Cocci
* SPHERICAL
Grouping
* DIPLOCOCCI - pairs, kidney-shaped
Bacteria:
Gram-Negative Rods/Bacillus
* RECTANGULAR shaped bacterium
Groupings:
* LONG, NARROW, SHORT CHAINS
* COCCOBACILLI - very short, narrow coccoid rods
* CURVED - very short, narrow & CURVED, short chains
* FUSIFORM - long, narrow, POINTED ends
* SPIRAL - long, narrow, MULTIPLE CURVES
Yeasts:
Blastoconidia
BUDDING yeasts
Yeasts:
Pseudohyphae
* ELONGATED PROJECTIONS from yeasts
* TAPERED ends
Artifacts:
Crystalline Precipitate (Crystal Violet Precipitate)
* BLUE to PURPLE
* May be REFRACTILE
* Formed in Crystal Violet Reagent over time
* May appear as BLUE AMORPHOUS material
* May resemble gram-positive cocci and rods
* May adhere to cells & tissue
Artifacts:
Cellular Debris
* Background debris
* MUCOUS STRANDS
* RUPTURED OR DISINTEGRATING CELLS
* PROTEIN PRECIPITATE
Artifacts:
Over-decolorization
* Gram-positive organisms partially appears like gram-negative bacteria
* May be due to POOR STAINING technique
* Critical during Acetone-Alcohol staining
* May occur if organisms are old or dead
* Due to damage to the organism's cell wall
Gram-negative Staining
1. EPITHELIAL CELLS
2. MUCOUS
3. WBCs
4. CELLULAR DEBRIS
Artifacts:
Antibiotic Effects
Alters the size, shape, and arrangement of some species of bacteria
Specimen Sites:
CSF
* Normally sterile
* Rare lymphocytes may be present
* PMNs are not normally present
Infection:
* Single morphotype of bacterium or yeast present
* Presence of PMNs &/or mononuclear cells
Specimen Sites - CSF:
Gram Positive Bacteria
1. Listeria monocytogenes
2. Group B Streptococcus
3. Streptococcus pneumoniae
Listeria monocytogenes
* SHORT, NARROW GRAM POSITIVE RODS
* Bacterial meningitidis & septicemia in neonates
* TUMBLING MOTILITY on wet mount
* UMBRELLA growth in motility agar
* Blood agar & hanging drop motility test
* Common in patients with LYMPHOMA
* Hydrolyzes esculin
Streptococcus pneumoniae
* GRAM POSITIVE DIPLOCOCCI, single, or short chains (elongated, LANCET shaped)
* With PMNs
* Bacterial MENINGITIDIS in adults
* Bacterial PNEUMONIA
* ALPHA HEMOLYSIS (greening of surrounding agar)
* BILE SOLUBLE on Blood agar
* Bacterial CONJUNCTIVITIS
* Round, translucent, glistening, dome-shaped when young
* Central depression with age
* Encapsulated strains are mucoid
* Normal in upper respiratory tract
* Community acquired pneumonia
* Meningitis in adults
* Infects sinuses, eyes
* OXACILLIN disk test
* QUELLING reaction
Specimen Sites - CSF:
Gram Negative Bacteria
1. Escherichia coli
2. Haemophilus influenzae
3. Neisseria meningitidis
Escherichia coli
* Long, narrow GRAM NEGATIVE RODS
* Bacterial meningitidis in infants
* Associated with upper UTI such as pyelonephritis
Haemophilus influenzae
* Small, pale, short & narrow, pleomorphic GRAM NEGATIVE RODS
* BACTERIAL MENINGITIS in children
* Presence of PMNs
* MOUSE NEST ODOR
* Requires X (hemin) & V (NAD) factors
Neisseria meningitidis
* Gram negative diplococci (kidney-shaped)
* Epidemic meningitidis
Specimen Sites - CSF:
Yeasts
1. Candida albicans
2. Cryptococcus neoformans
Candida albicans
* BUDDING YEASTS
* PSEUDOHYPHAE with tapered ends
* Meningitidis
* Cause ulcerative lesions of the intestines in immunocompromised patients
* Important cause of hospital acquired intravenous catheter infections
* Isolated from urine of patients on antibiotics or diabetic patients
Cryptococcus neoformans
* Encapsulated yeasts of various sizes
* Chronic meningitidis in immunocompromised patients
Specimen Sites:
Blood
Septicemia
* Gram positive diplococcus
Contamination
* Gram positive cocci in clusters
Specimen Site - Blood:
Gram Positive Bacteria
1. Clostridium perfringens
2. Clostridium tertium
3. Corynebacterium spp
4. Enterococcus faecalis
5. Lactobacillus spp
6. Listeria I spp
7. Micrococcus I spp
8. Propionibacterium spp
9. Staphylococcus spp
10. Streptococcus mitis
11. Streptococcus pneumoniae
Clostridium perfringens
* Large gram positive rods w/blunt ends
* Gas gangrene
* Box-car shaped
* Double zone of beta hemolysis on blood agar
Clostridium tertium
* Gram positive rods
* Aerotolerant Clostridium species
Corynebacterium spp
* Gram positive rods grouped adjacently
* Normal skin inhabitants that can cause intravenous catheter infections
Enterococcus faecalis
* Gram positive cocci in chains
* Normal intestinal flora
* Associated with intraabdominal infections
* Usually nonhemolytic
* May be alpha or beta-hemolytic
* Causes nosocomial UTI, wound infections, bacteremia
* Hydrolyzes esculin
* Grows in 6.5% NaCl broth
* PYR positive
* Lancfield group D
* Similar to E. faecium
* Resistant to penicillin & some to Vancomycin (VRE)
Lactobacillus spp
* Gram positive rods (long, narrow)
* Isolated in urine specimens with vaginal contamination
Listeria I spp
* Gram positive rods (short, narrow)
* Important cause of meningitidis in neonates & immunocompromised patients
Micrococcus I spp
* Gram positive cocci in pairs, tetrads, & clusters
* Resemble Staphylococcus on gram's stain
* Generally considered contaminants
* Often pigmented (yellow, orange, tan)
* Usually non-pathogenic
* Foundin environment & on skin
* CATALASE positive
* COAGULASE NEGATIVE
* Oxidative, Modified oxidase-positive
* Microdase-positive
* Sensitive to bactracin & lysozyme
* Resistant to furazolidone
Propionibacterium spp
* Gram positive rods (pleomorphic)
* Members of the skin flora
* Rarely cause prosthetic device infections
Streptococcus mitis
* Gram positive cocci (chains)
* Causes endocarditis
Specimen Site - Blood:
Gram Negative Bacteria
1. Escherichia coli
2. Klebsiella pneumoniae
3. Pseudomonas spp
Klebsiella pneumoniae
* Gram negative rods
* Cause of nosocomial pneumonia
Pseudomonas spp
* Gram negative rods
* Cause of burn & wound infections
Specimen Site - Blood:
Yeasts
Candida albicans
Specimen Site - Respiratory
Contaminated Sputum
* Contamination of saliva
* Abundant squamous epithelial cells & very few PMNs
Acceptable Sputum
* Very few squamous epithelial cells
Specimen Site:
Respiratory
1. Pneumonia
Pneumonia
* Ciliated columnar epithelial cells from the lower respiratory tract
* Large number of PMNs & bacteria in patients with bacterial pneumonia
Specimen Site - Respiratory:
Gram Positive Bacteria
1. Corynebacterium spp
2. Nocardia asteroides
3. Staphylococcus aureus
4. Streptococcus pneumoniae
Corynebacterium spp
* Gram positive rods grouped adjacently/pleomorphic
* Opportunistic pathogens
* Cause pneumonia in immunocompromised patients
* Squamous epithelial cells
Nocardia asteroides
* GRAM POSITIVE BRANCHED, BEADED RODS with fragmentation
* Soil bacteria
* Cause PNEUMONIA in immunocompromised patients
* Beta hemolytic
* Wrinkled
* Dry
* Crumbly
* Chalky white to orange tan
* Brain abscess
* ACID FAST utilizing a weak decolorizing agent
Staphylococcus aureus
* Gram positive cocci in clusters, tetrads & gram negative coccobacillus (MIXED BACTERIAL INFECTIONS)
* Causes pneumonia in hospitalized patients with other pulmonary infections
* Common in WOUND specimens
* Causes EYELID & CORNEAL infections
* Common cause of bacterial ARTHRITIS
* Yellow colonies & beta-hemolytic (small zone)
* Causes suppurative cutaneous infections, toxic shock syndrome, food poisoning
* Coagulase positive
* Ferments mannitol
* DNAse positive
* 85-90% penicillin resistant
Specimen Site - Respiratory:
Gram Negative Bacteria
1. Haemophilus infulenzae
2. Klebsiella pneumoniae
3. Moraxella catarrhalis
4. Neisseria meningitidis
5. Pseudomonoas aeruginosa
Haemophilus infuenzae
* Small, pale, pleomorphic gram negative rods
* Cause pneumonia in patients with underlying respiratory disease
Klebsiella pneumoniae
* Gram negative rods (long, narrow)
* Leading cause of hospital acquired bacterial pneumonia
Moraxella catarrhalis
* Gram negative diplococci
* Cause pneumonia in patients with emphysema or asthma
* Intracellular bacteria
* White blood cell (PMNs)
* Extracellular bacteria
* HOCKEY PUCK colonies
Neisseria meningitidis
* Gram negative diplococci
* Rare cause of acute bacterial pneumonia
* Intracellular bacteria
* White blood cell (PMNs)
* Glucose & Maltose positive
Pseudomonas aeruginosa
* LONG, NARROW GRAM NEGATIVE RODS
* Common cause on pneumonia in CYSTIC FIBROSIS patients
* Infects burn sites
* Associated with HOT TUB FOLLICULITIS
* Leads to blindness
* Grapelike odor
Specimen Site - Respiratory:
Yeasts
Candida albicans
Specimen Site:
Urine
Normal
* Rare epithelial cells may be present
* PMNs are not normally present
Infection
* PMNs present = UTI
* Single bacterium per oil immersion indicated infection with approximately 100,000 organisms per mL of urine
Contaminated
* Bacteria from periurethral area
* Squamous epithelial cells & mixture of gram positive organisms
* Gram negative rods may be present
Specimen Site - Urine:
Gram Positive Bacteria
1. Enterococcus faecalis
2. Staphylococcus saprophyticus
Enterococcus faecalis
* Gram positive cocci
* Causes hospital acquired UTI
Staphylococcus saprophyticus
* Small CLUSTERS & PAIRS of gram positive cocci
* Common cause of UTI in young females & catheterized elderly men
* CHALKY WHITE to slightly yellow
* NOVOBIOCIN RESISTANT
* Blood agar
* Nonhemolytic
* Coagulase negative
* May ferment mannitol
Specimen Site - Urine:
Gram Negative Bacteria
1. Escherichia coli
2. Klebsiella spp
3. Proteus spp
4. Pseudomonas spp
Klebsiella spp
* Long, narrow gram negative rods
* Associated with the formation of kidney stones
Proteus spp
* Long, narrow gram negative rods
* P. mirabilis - associated with the formation of kidney stones
Pseudomonas spp
* Long, narrow gram negative rods
* P. aeruginosa - common cause of hospital acquired UTI
Specimen Site - Urine:
Yeasts
Candida albicans
Specimen Site - Genital
Normal Vagina
* Commonly see squamous epithelial cells
* Normal flora - Lactobacilli
* Presence of occasional PMNs
Vaginosis
* Clue cells - squamous epithelial cells with large numbers of adherent coccobacilli
* CLUE CELLS = Bacterial Vaginosis
* Gram variable coccobacilli
Urethritis (males)
* Intracellular gram negative diplococci = GONORRHEA
* Commonly see PMNs
Specimen Site - Genital:
Gram Variable Bacteria
1. Gardnerella vaginalis
2. Mobiluncus curtisii
3. Neisseria gonorrhea (female)
4. Neisseria gonorrhea (male)
Gardnerella vaginalis
* Gram positive or gram negative coccobacilli
* Vaginal specimen associated with BACTERIAL VAGINOSIS
* CLUE CELL with many ADHERENT gram variable bacteria
* Fish like odor
Mobiluncus curtisii
* Gram variable short, curved rods
* Associated with bacterial vaginosis
Neisseria gonorrhea (female)
* Gram negative diplococci, coffee bean shaped
* White blood cell PMNs
* Squamous epithelial cell with adherent bacteria
* Extracellular bacteria
Neisseria gonorreha (male)
Intracellular GRAM NEGATIVE DIPLOCOCCI (coffee bean shaped) associated with GONORRHEA
Specimen Site - Genital:
Parasites
1. Trichomonas vaginalis
Trichomonas vaginalis
* Oval shaped
* Can resemble a white blood cell or a small epithelial cell
* Causes vaginitis
Specimen Site - Wound
Not Infected
* normal flora in non-sterile site
* no bacteria in sterile site
* presence of epithelial cells
Infected
* Large numbers of PMNs
* Wide variety of bacterial species
* Caused by a mixture of bacteria
Specimen Site - Wound:
Gram Positive Bacteria
1. Actinomyces spp
2. Clostridrium perfringens
3. Enterococcus spp
4. Staphylococcus aureus
5. Staphylococcus epidermidis
6. Group A Streptococcus
Actinomyces spp
* Branched gram positive rods
* Cause wound infection involving the mouth or jaw
* Club-shaped
* Molar tooth colonies
Clostridum perfringens
* Long, wide gram positive rods
* Cause gas gangrene
Enterococcus spp
* Gram positive cocci
* Normal fecal flora
* Commonly cause abdominal would infections
Staphylococcus epdermidis
* Gram positive cocci in clusters
* Part of the normal skin flora
* Cause opportunistic infections
* Associated with indwelling catheter infections
Group A Streptococcus
* S. pyogenes
* Gram positive cocci in chains
* Cause wound infections in a variety of sites
* PINPOINT colonies
* Grayish white, translucent
* Usually beta hemolytic
* Cause bacterial arthritis
* Cause 90% of strep infections
* Strep sore throat, rheumatic fever, glomerulonephritis, scarlet fever (scarlatina), erysipelas, puerperal sepsis, impetigo
* Sensitive to bacitracin
* Resistant to SXT
* PYR positive
* Susceptible to penicillin
Specimen Site - Wound:
Gram Negative Bacteria
1. Bacteroides spp
2. Eikenella corrodens
3. Pasteurella multocida
4. Proteus mirabilis
5. Pseudomonas aeruginosa
6. Serratia marcescens
Bacteroiides spp
* VARIABLE length, pale staining, gram negative rods
* Cause intra-abdominal abscesses
Eikenella corrodens
* Long, narrow, gram negative rods
* Cause soft tissue abscesses including brain abscess
Pasteurella multocida
* Gram negative coccobacilli rods w/bipolar staining
* Commonly found in wound infections resulting from animal bites
* Musty odor
Proteus mirabilis
* Long, narrow gram negative rods
* Cause infections in burn sites
Serratia marcescens
* LONG, NARROW, SPIRAL GRAM NEGATIVE RODS
* Abundant CELLULAR DEBRIS
* Presence of PMNs
* Cause wound infections associated with trauma or surgery
Specimen Site - Wound:
Mixed Bacteria
* Isolated from abnormal abscesses
1. Fusiform & Spiral
Fusiform & Spiral
* Cause ulcerative dental infection - Trench Mouth
Specimen Site - Eye
* Normal flora - conjunctiva colonized with nonpathogenic corynebacteria & staphylococci from skin with rare PMNs
* Infection is present with many PMNs & one morphotype of bacteria are seen
1. Staphyloccocus aureus
2. Streptococcus pneumoniae
3. Haemophilus spp
4. Moraxella spp
5. Pseudomonas aeruginosa
Haemophilus spp
* Small, pale, pleomorphic gram negative rods
* Conjunctivitis epidemics in day care centers
Moraxella spp
* Gram negative cocci
* Causes bacterial conjunctivitis
Specimen Site - Joint Fluids
* Infection with presence of PMNs & single morphotype of bacteria
Gram positive
1. Staphyloccus aureus
2. Group A Streptococcus
Gram negative
1. Haemophilus influenzae
2. Neisseria gonorrhea
Haemophilus influenzae
* Small, pale, pleomorphic gram negative rods
* Cause bacterial arthritis in children
Neisseria gonorrhea
* Gram negative diplococci (coffee bean shaped)
* Bacterial arthritis due to gonococci during systemic gonococcal disease
Specimen Site - Stool
Infection
* PMNs - invasive pathogen is present
* Reduction of normal fecal flora - Diarrhea
* Usually gram negative rods
Gram positive
1. Clostridium difficile
Gram negative
1. Campylobacter spp
2. Salmonella spp
3. Shigella spp
Yeasts
1. Candida albicans
Parasites
1. Cryptosporidium parvum
Clostridium difficile
* Long, wide, gram positive rods with spores
* Causes antibiotic diarrhea & pseudomembranous colitis in hospitalized patients
* Yellow ground-glass colonies
Campylobacter spp
* Very short, narrow, CURVED GRAM NEGATIVE RODS
* Normally carried by animals & can CAUSE BLOOD DIARRHEA in humans
* GULL WING morphology
Salmonella spp
* Long, narrow, gram negative rods
* Carried by poultry
* Cause food poisoning
Shigella spp
* Long, narrow gram negative rods
* Highly infectious human pathogen
* Causes bloody, mucoid diarrhea (dysentery)
Cryptosporidium parvum
* Variably staining, circular or oval shaped
* Cause diarrhea in immunocompromised patients
Neonatal meningitis
1. Group B Streptococcus
2. Enterobacteriaceae
3. Listeria
Group B Streptococcus
* S. agalactiae
* Blood Agar
* BETA HEMOLOYSIS (clearing of the agar) around each of the colonies
* GRAM POSITIVE COCCI IN CHAINS
* Bacterial meningitidis & septicemia in neonates
* Semi opaque
* Bull's eye colonies
* Occasionally non-hemolytic
* Normal flora of female genital tract
* Resistant to SXT & bacitracin
* SODIUM HIPPURATE & CAMP POSITIVE
Hemolysis on Agar
1. Certain Streptococcus
2. Staphylococcus aureus
3. Listeria
Clostridium septicum
* LONG, WIDE GRAM POSITIVE RODS
* Associated with MALIGNANCY (CANCER)
Associated with Malignancy
1. Clostridium septicum
2. Streptococcus bovis
Organisms in CO2
1. Campylobacter
2. Haemophilus
3. Helicobacter
4. Moraxella catarrhalis
5. Mycobacterium
6. Pathogenic Neisseriae
Media for Aerobes & Facultative Anaerobes
1. Sheep Blood Agar
2. Chocolate Agar
3. Columbia Colistin-Nalidixic Acid Agar (CNA)
4. Phenylethyl Alcohol Agar (PEA)
5. Streptococcal Selective Agar (SSA)
6. Eosin Methylene Blue (EMB)
7. MacConkey Agar (MAC)
8. MacConkey Sorbitol Agar (SMAC)
9. Hektoen Enteric Agar (HE)
10. Xylose Lysine Deoxycholate (XLD)
11. Salmonella-Shigella Agar (SS)
12. Gram-Negative Broth (GN)
13. Selenite Broth
14. Tetrathionate Broth
15. Campylobacter Blood Agar (Campy BA)
16. Modified Thayer-Martin (TM)
17. Martin-Lewis
18. New York City Medium (NYC)
Sheep Blood Agar
Allows differentiation of hemolysis
Chocolate Agar
* Haemophilus & Neisseria
* Supplies X & V factors
Columbia Colistin-Nalidixic Acid Agar (CNA)
* Gram-Positives
* Suppress most gram-negatives
Phenylethyl Alcohol Agar (PEA)
* Gram-Positive cocci & Anaerobic gram-negative rods
* Inhibits gram-negative rods
Streptococcal Selective Agar (SSA)
S. pyogenes & S. agalactiae
Eosin Methylene Blue (EMB)
* Enteric gram-negative rods
* Inhibit gram-positives
* Lactose fermenters = green-black or purple
* E. coli = green metallic sheen
* Non-lactose fermenters = colorless
MacConkey Agar (MAC)
* Isolation of enteric gram-negative rods
* Inhibit most gram-positives
* Lactose fermenters = pink
* Non-lactose fermenters = colorless
MacConkey Sorbitol Agar (SMAC)
E. coli O157:H7 does not ferment sorbitol = colorless
Hektoen Enteric Agar (HE)
* Isolation of enteric pathogens from stool
* Inhibit normal GI flora
* Non-pathogens = orange to salmon pink
* Non-lactose fermenters = green to blue-green
* H2S-positive colonies = black precipitate
Xylose Lysine Deoxycholate (XLD)
* Isolation of Salmonella & Shigella from stool
* Inhibits many gram-negative rods & gram-positives
* E. coli = yellow
* Some Proteus spp = yellow with black centers
* Shigella = colorless or red
* Salmonella = red with black centers
Salmonella-Shigella Agar (SS)
* Selective for Salmonella & Shigella
* Inhibit coliforms
* Do not ferment lactose = colorless
* Salmonella = produces H2S with black center
Gram-Negative Broth (GN)
* Isolation of Salmonella & Shigella from stools & rectal swabs
* Retard growth of gram-positives
Selenite Broth
Recovery of Salmonella from stool
Tetrathionate Broth
* Recovery of Salmonella from stool except S. typhi
* Inhibit gram-positives & Enterobacteriaceae
* Inhibits most Shigellae
Campylobacter Blood Agar (Campy BA)
Isolation of Campylobacter from stool
Modified Thayer-Martin (TM)
* Recovery of Neisseria gonorrhoeae & Neisseria meningitidis from specimens with normal flora
* Inhibit growth of other bacteria & fungi & some N. gonorrhoeae
Martin-Lewis
* Recovery of N. gonorrhoeae & N. meningitidis from specimens with normal flora
* Inhibits yeast better
New York City Medium (NYC)
* Isolation of N. gonorrhoeae & N. meningitidis from specimens with normal flora
* Some N. gonorrhoeae are inhibited by antibiotics
* Growth of genital mycoplasmas
Media for Anaerobes
1. Bacteroides Bile-Esculin Agar (BBE)
2. Anaerobic Blood Agar, CDC
3. Cycloserine Cefoxitin Fructose Egg Yolk Agar (CCFA)
4. Cooked Meat Medium
5. Egg-Yolk AGar (EYA)
6. Laked Kanamycin-Vancomycin Blood Agar (LKV)
7. Phenylethyl Alcohol Agar (PEA)
8. Thioglycollate Broth (THIO)
Bacteroides Bile-Esculin Agar (BBE)
* Isolation & Identification of Bacteroides fragilis
* Hydrolysis indicated by blackening of agar
Anaerobic Blood Agar,CDC
Isolation of fastidious anaerobes
Cycloserine Cefoxitin Fructose Egg Yok Agar (CCFA)
Selective for Clostridium difficile
Cooked Meat Medium
Isolation of anaerobes, especially pathogenicClostridium
Egg-Yolk Agar (EYA)
Determination of lecithinase & lipase production by clostridia & fusobacteria
Laked Kanamycin-Vancomycin Blood Agar (LKV)
Selective for Bacteroides & Prevotella
Phenylethyl Alcohol Agar (PEA)
Inhibits enteric gram-negative rods & swarming by some clostridia
Thioglycollate Broth (THIO)
* Supports the growth of most aerobes & anaerobes
* Backup broth to detect organisms present in small numbers or anaerobes
* Reducing agent
* Aerobes = grow at top
* Anaerobes = grow at bottom
* Facultative anaerobes = grow throughout
Special Bacteriologic Media
1. Cystine-Tellurite Blood Agar
2. Loeffler Medium
3. Tindale Agar
4. Bismuth Sulfite Agar
5. Cefsulodin-Irgason-Novobiocin Agar
6. Alkaline Peptone Water
7.Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS)
8. Rabbit Blood Agar
9. Bordet-Gengou
10. Regan Lowe
11. Buffered Charcoal-Yeast Extract Agar (BCYE)
12. Vaginalis Agar (V Agar)
13. Human Blood Tween Agar (HBT)
14. Lowenstein-Jensen
15. Middlebrook 7H10 & 7H11
16. Fletcher Medium
Cystine-Tellurite Blood Agar
Isolation of Corynebacterium diphtheriae = black colonies
Loeffler Medium
* Recovery & identification of C. diphtheriae
* Promotes development of characteristic granules
Tindale Agar
* Isolation & identificationof C. diphtheriae = colonies surrounded by a brown halo
* Gray to black colonies due to reduction of tellurite
Bismuth Sulfite Agar
* Isolation of Salmonella = light green
* S. typhi = black colonies surrounded by a metallic sheen
Cefsulodin-Irgason-Novobiocin Agar (CIN)
* Selective for Yersinia enterocolitica, Aeromonas, & Plesiomonas shigelloides
* Crystal violet = inhibits most gram-negatives
* Novobiocin = inhibits gram-positive cocci
* Cefsulodin = inhibits most gram-positives & gram-negatives
* Y. enterocolitica = ferments mannitol, bull's eye colonies surrounded by colorless halo
Alkaline Peptone Water
* Recovery of Vibrio &b Aeromonas from stool
* Alkaline pH suppresses commensals
* Subcultured to TCBS
Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS)
* Selective for Vibrios
* High pH inhibits most bacteria
* V. cholerae = ferments sucrose & produces yellow colonies
* V. parahaemolyticus & V. vulnificus don't ferment sucrose & usually produce blue-green colonies
Rabbit Blood Agar
* Speciates Haemophilus
* H. haemolyticus & H. parahaemolyticus = hemolytic
Bordet-Gengou
* Isolation of Bordatella pertussis = mercury droplets
* Contaminants inhibited by methicillin
* Cough plate
Regan Lowe
Selective for B. pertussis
Buffered Charcoal-Yeast Extract Agar (BYCE)
* Isolation of Legionella
* Yeast extract & l-cysteine enhance growth of Legionella
* Charcoal absorbs toxic compounds
Vaginalis Agar (V Agar)
Isolation of Gardnerella vaginalis = diffuse beta hemolysis
Human Blood Tween Agar (HBT)
Semi-selective for G.vaginalis = diffuse beta hemolysis
Lowenstein-Jensen
* Culture mycobacteria
* Malachite green inhibits other bacteria
* M. tuberculosis = rough & buff
Middlebrook 7H10& 7H11
* Culture mycobacteria
* Agar-based
* Isoniazid-resistant strains grow better than on egg-based media
* Malachite green = inhibit other bacteria
Fletcher Medium
Isolation of Leptospira from blood, CSF, & urine
Gram-Positive Cocci
1. Staphylococcus spp
2. S. aureus
3. S. epidermidis
4. S. saprophyticus
5. Micrococcus
6. Streptococcus spp
7. Group A streptococci
8. Group B streptococci
9. Group D streptococci, nonenterococci
10. Enetrococcus
11. Streptococcus pneumoniae
12. Viridans streptococci
Staphylococcus spp
* Gram-positive cocci, usually in clusters
* Facultative anaerobe
* Blood agar = 1-3mm, round, smooth, convex, glistening, opaque, entire edge, butyrous
* Normal flora of skin, mouth, pharynx, vaginal, urethra, GI tract
* Catalase positive
* Fermentative
* Resistant to bactracin & lo
S. epidermidis
* Gram-positive cocci, usually in clusters
* White colonies, usually nonhemolytic
* Usually non-pathogenic
* Normal on cutaneous & mucosal surfaces
* Most infections are hospital-acquired
* Common cause of nosocomial UTI
* Coagulase negative
* Grows on mannitol salt agar but doesn't ferment mannitol
* Sensitive to novobiocin
Streptococcus spp
* Gram positive cocci, somewhat elongated, in chains, & in pairs
* Facultative anaerobe
* Require enriched media
* CATALASE NEGATIVE
* <1mm, white to gray, translucent, or semiopaque
* Variable hemolysis
Group D stretococci, nonenterococci
* Gram positive cocci in chains
* Slightly opaque
* Usually nonhemolytic
* May be alpha hemolytic
* Normail in GI tract
* Causes nosocomial UTI, wound infections, bacteremia
* HYDROLYZES ESCULIN
* S. bovis & S. equinus
Viridans streptococci
* Gram positive cocci in chains
* Normal in oral respiratory, GI mucosa
* Opportunistic pathogens
* Frequent cause of subacute bacterial endocarditis
* Dental caries
* OPTOCHIN RESISTANT
* NON BILE SOLUBLE
Gram Positive Tests
1. Catalase
2. Coagulase
3. Mannitol Salt Agar
4. Novobiocin
5. Bacitracin Disk
6. SXT
7. PYR
8. CAMP Test
9. Hippurate Hydrolysis
10. Bile Esculin Agar
11. 6.5% NaCl Broth
12. Optochin Disk (P Disk)
13. Bile Solubility
14. Slide Agglutination Test
Catalase
* Staphylococci
* Enzyme catalase converts 3% hydrogen peroxide to oxygen & water, resulting in immediate bubbling
Coagulase
* S. aureus
* Enzyme coagulase causes coagulation (tube reflex test) or agglutination (slide screening test) in rabbit plasma
Mannitol Salt Agar
* S. aureus
* Mannitol fermentation = pink to yellow
Novobiocin
* S. saprophyticus
* Resistant = growth at the edge of the disk
* Performed on coagulase negative staph isolated from urine
Bacitracin disk
* Group A strep
* Zone of inhibition = organism is susceptible to bactracin
SXT
* Group A & B resistant = growth up to the disk
* Differentiates Group A strop from Group C or G strep
PYR
* Group A strep & enterococci
* Red color
CAMP test
* Group B strep
* Arrowhead formation
* Enhances hemolysis of beta hemolytic S. aureus
Hippurate hydrolysis
* Group B strep
* Precipitate formation upon addition of ferricchloride
Bile Esculin Agar
* Group D strep & enterococci
* Dark brown
* Inhibits gram positive bacteria
6.5% NaCl Broth
* Enterococci
* Turbidity
Optochin Disk (P Disk)
* S. pneumoniae
* Zone of inhibition >/ 14mm w/a 6mm disk
* Differentiate S. pneumoniae from viridans strep
Bile solubility
* S. pneumonia
* Causes lysis
Slide Agglutination Tests
* Rapid identification of S. aureus, streptococci (Group A,B,C,D,F,G), & S. pneumoniae
* Reagent antibodies react with bacterial antigens = agglutination
Gram Positive Bacilli
1. Bacillus spp
2. Bacillus anthracis
3. Corynebacteria spp
4. Erysipeelothrix
5. Lactobacillus
6. Listeria monocytogenes
7. Nocardia
Bacillus anthracis
* Box Cars
* Bamboo
* Medusa head colonies
Corynebacteria
* Chinese letter or palisade formation (diphtheroid arrangement)
* Black colonies with brown halos on modified Tindale's agar
* Black or gray colonies on Cystine-Tellurite Blood Agar
* Catalase Positive
Erysipelothrix
* PINPOINT
* Filaments,short chains, Vs
* Nonhemolytic or alpha hemolytic
* Catalse negative
* Non motile
* H2S Positive
* Indole Negative
* Resistant to neomycin
Lactobacillus
* Flora of vagina during reproduction
* Pinpoint alpha hemolytic, rough gray colonies
* Catalase negative
Lactose Negative
1. Citrobacter
2. Edwardsiella
3. Morganella
4. Proteus
5. Providencia
6. Salmonella
7. Serratia
8. Shigella
9. Yersinia
H2S Positive
1. Citbrobacter
2. Edwardsiella
3. Proteus
4. Salmonella
VP Positive
1. Enterobacter
2. Klebsiella
3. Serratia
PD Positive
1. Morganella
2. Proteus
3. Providencia
Urease Positive
1. Klebsiella
2. Morganella
3. Proteus
4. Providencia rettgeri
Nonmotile at 35C
1. Klebsiella
2. Shigella
3. Yersinia (motile at 22C)
TSI A/A
1. Serratia
2. Escherichia
3. Enterobacter
4. Klebsiella
TSI A/A, H2S+
1. Citrobacter
2. Arizona
3. Proteus
TSI K/A, H2S+
1.Citrobacter
2. Arizona
3. Salmonella
4. Edwardsiella
TSA K/A
1. Shigella
2. Citrobacter
3. Providencia
4. Proteus
5. Yersinia
TSA K/K
Pseudomonas
Haemophilus ducreyi
* Requires X (hemin) Factor
* Chancroid
* School of Fish
Bartonella
* Trench fever
* Cat-scratch disease
Bordatella
Whooping cough in children & adults
Brucella
* Brucellosis (undulant fever)
* From unpasteurized milk or contact with infected goats, cows, hogs, dogs
Francisella
* Tularemia (Rabbit Fever)
* Man infected by tick or handling infected animal
Clostridium tetani
* Gram positive rods with swollen terminal spores
* Drumstick
* Tennis racquet
* Becomes gram negative after 24hrs
Propionibacterium
* Causes acne
* Catalase positive
* Indole positive
* Pleomorphic gram positive rods
* Diphtheroidlike
Bacteroides fragilis
* Gram negative, pleomorphic, pale staining rods
* Bipolar staining
* Safety pins
* Resistant to bile
* Hydrolyses esculin
* Turnes BBE brown
* Resistant to kanamycin
Anaerobes
1. Gram positive Cocci:
*Peptostreptococcus
2. Gram Positive Bacilli:
* Actinomyces
* Clostridium difficile
* Clostridium perfringens
* Clostridium tetani
* Propionibacterium
3. Gram Negative Cocci
* Veillonella
4. Gram Negative Bacilli
* Bacteroides fragilis
* Fusobacterium
* Prophyromonas
* Prevotella
Acid Fast Stains
1. Ziehl-Neelsen
2. Kinyoun
3. Fluorochrome
Ziehl-Neelsen
1. Carbolfuchsin - primary stain
2. Acid Alcohol - decolorizer
3. Methylene blue - counterstain
* red
* slightly curved
* beaded rods (2-8um)
* blue background
* requires heat
* examine at least 300 oil immersion fields
Kinyoun
1. Carbolfuchsin - primary stain
2. Acid alcohol - decolorizer
3. Methylene blue - counterstain
* red
* slightly curved
* beaded rods (2-8um)
* blue background
* cold stain
* examine at least 300 oil immersion fields
Fluorochrome
1. Auramine-rhodamine or auramine O - primary stain
2. Acid alcohol - decolorizer
3. Potassium permanganate or acridine orange - counterstain
* yellow, orange, or red rods against dark background
Spirochetes
1. Borrelia recurrentis
2. Borrelia burgdorferi
3. Treponema pallidum pallidum
4. Treponema pallidum pertenue
5. Treponema pallidum endemicum
6. Treponema carateum
7. Leptospira interrogans
Borrelia recurrentis
* Relapsing fever
* Grows on modified Kelly medium
* Transmitted by ticks or lice
Borrelia burgdorferi
* Lyme disease (erythema chronicum migrans, neurological & cardiac abnormalities, arthritis)
* Grows on modified Kelly medium
* Serology is most common method
* Tick-borne infection - Ixodes tick
Treponema pallidum pallidum
* Syphilis
* Darkfield microscopy
T. pallidum pertenue
Yaws (skin lesions & bone destruction)
T. pallidum endemicum
Bejel (endemic syphilis)
T. carateum
Pinta (skin lesions, cardiovascular & nervous system involvement)
Leptospira interrogans
* Weil's disease (infectious jaundice)
* Grows on Fletcher or Stuart media
Mycoplasma pneumoniae
* Fried egg appearance
* Smallest free-living cell
* Require medium enriched with cholesterol & fatty acids
Mycoplasma hominis & Ureaplasma urealyticum
* Urogenital tract disease
* Less stringent in growth requirements but require cholesterol for synthesis of plasma membranes
Rickettsia
* Rocky Mountain spotted fever
* Rickettsialpox
* Epidemic typhus
* Murine typhus
* Scrub typhus
* Grow in lice, ticks, tissue culture, eggs
Coxiella
* Q fever
* Cell culture (lung fibroblasts)