Practical 2

Catalase Test
test used to distinguish Staphylococcus from Streptococcus and Enterococcus; if Staph, produces enzyme called catalase that reacts with toxic oxygen by-product hydrogen peroxide, breaking into water and molecular oxygen. Bubbling reaction will occur if positve
Lancefield Classification
categorizes streptococci based on similar surface antigens
Streptococcus Identification
negative catalase test and inability to of most species to grow in presence of high salt; are capnophiles, produce hemolysins, susceptible to antibiotics bacitracin and SXT, and ability to hydrolyze esculin in bile esculin slants
grow best in presence of carbon dioxide
hydrolyze red blood cells
Beta hemolysis
complete lysis of red blood cells and hemoglobin; results in complete clearing of blood around colonies
We will write a custom essay sample on
Any topic specifically for you
For only $13.90/page
Order Now
Alpha hemolysis
partial lysis of red blood cells and hemoglobin; results in greenish-grey discoloration of blood around the colonies
Gamma hemolysis (no hemolysis)
no change in medium
Mannitol Salt Agar (MSA) for Strep ID
selective and differential; solely used as selective in Strep id; contains 7.5% NaCl (selects for halotolerant organisms); only strep family able to grow on MSA is Enterococcus species
Blood Agar
differential medium; commonly used as enriched medium for growing fastidious bacteria; if bacteria produce exotoxins called hemolysins will cause lysis of red blood cells in agar; useful for id of strep species depending on degree of hemolysis; also used to test susceptibility to bacitracin and SXT
Bile Esculin Agar Slant
Selective and differential; contains bile salts, esculin, ferric ammonium citrate, and beef and gelatin extracts;
Bile Salts in BEA
give BEA its selective property; only enteric organisms are able to grow in presence of bile
Esculin in BEA
BEA differential property; distinguishes organisms able to hydrolyze carbohydrate esculin; esculin is hydrolyzed to glucose and esculetin (reacts with ferric ammonium citrate)
Ferric ammonium citrate in BEA
BEA differential property; esculetin (hydrolyzed from esculin) reacts with ferric ammonium citrate to form blackish precipitate
Beef and gelatin extracts in BEA
provide macromolecules for general growth requirements
Susceptibility to Bacitracin and SXT
SXT (trimethoprim-sulfamethoxazole) on blood plate: zone of inhibition os SXT disc (greater than 20mm = susceptible), Any zone of inhibition with bacitracin disk = susceptible
Rapid Strep Test
allows for quick diagnosis of Group A Strep directly from throat swabs; uses a sandwich immunoassay to detect group A strep antigens; test cassette has membrane strip with anti-Step A antibodies; if specimen is GAS Strep A antigens bind to antibodies (antigen-antibody complex); binds with 2nd antibody conjugated with chromogen (produces colored line). Positive = red line in both test and control line; Negative = only have a red line in control region
Gram +; classified into 2 major groups: S. aureus and non-S. aureus;
S. aureus
leading cause of skin and soft tissue infections (SSTIs), Toxic Shock Syndrome (TSS), and scalded skin syndrome; found to be causative agent in ailments such as pneumonia, meningitis, arthritis, and osteomyelitis; has ability to ferment Mannitol, positive result in coagulase test
methicillin-resistant S. aureus; resistant to penicillin-derived methicillin and other antibiotics; emerged in hospitals; significant threat to public health; CA-MRSA is community-acquired mrsa- not hospital associated, causing serious infections in healthy people
non-S. aureus
S. epidermidis- most clinically significant; opportunistic pathogen, normal resident of human skin; often infects I.V. drug users, newborns, elderly, and those using catheters or artificial appliances
ID of Staph species
can be distinguished from other gram + cocci by two main properties: 1) ability to grow on high salt media and 2) production of enzyme catalase; staph species distinguished from one another based on: 1) ability to ferment mannitol, 2) susceptibility to antibiotic Novobiocin and 3) whether or not produce hydrolytic enzyme called DNase (degrades DNA)
Mannitol Salt Agar (MSA) for Staph ID
selective and differential; 7.5% NaCl (halotolerant organisms); contains mannitol and phenol red (differentiation based on whether or not able to ferment mannitol); if mannitol fermentation, phenol red pH indicator changes color from red to yellow (due to acidic fermentation products reacting with pH indicator)
DNase agar
contains emulsion of DNA, peptides, and methyl green dye; dye and polymerized DNA form complex (gives agar blue-green color at pH 7.5); if bacterial colonies secret DNase, hydrolyze DNA in medium into smaller fragments; results in clearing around bacterial growth
Mueller-Hinton agar
nutrient agar used in antimicrobial testing; allows for antibiotic diffusion into agar; tests susceptibility to antibiotic Novobiocin; levels of thymine, thymide, calcium ions, and magnesium ions controlled in medium so as not to interfere with susceptibility testing; susceptible organisms will not grow adjacent to to disc; zone of inhibition greater than 18mm susceptible to Novobiocin, greater than 18mm resistant to novobiocin
Coagulase Test (DEMO)
allows differentiation of staph aureus from other staph strains (most staph aureus strains produce enzyme coagulase); coagulase acts within host tissue to convert fibrinogen to fibrin; Tube Test performed- s. aureus inoculated into tube of citrated plasma and incubated for 4 hours; clot forms within 4 hours positive for presence of coagulase (virulent S. aureus); tube test more sensitive than slide test; clots allow bacteria to “hide” from immune system
Rapid Staph Test
contains latex particles coated with human fibrinogen and IgG; latex particle bind with any staph bacteria that contain bound coagulase and/or Protein A, resulting in visible clumping or agglutination of latex particles; positive result = conclusive for staph aureus (replaces need to grow bacteria on selective and differential media)
Gram Negative bacteria
posses thin layer of peptidoglycan in cell wall plus outer phospholipid bilayer membrane containing lipopolysaccharide (LPS); found in places like soil, fresh water, and salt water; found in abundance in normal microbiota in intestinal tracts of mammals; constitute largest group of human bacterial pathogens (almost all has capacity to cause morbidity and mortality in humans), due in part to lipid A of LPS (endotoxin- fever, vasodilation, inflammation, shock, and blood clots within blood vessels
Identification of Gram-Negative species
constitutes 8 tests: MacConkey Agar, Triple-Sugar Iron Test, Urease Test, SIM agar test, MRVP Test, Citrate Utilization Test, Nitrate Reduction Test, and Lysine Iron Agar Test
MacConkey Agar
selective and differential; contains crystal violet and bile salts (inhibit most gram positives and select for gram negatives); contains carbohydrate substrate lactose, and pH indicator neutral red (allows differentiation among gram negative bacteria based on ability to ferment lactose; if lactose fermentation- acid-end products lower pH of media below 6.8, turn colonial growth pinkish-red; no lactose fermentation- colonies will be colorless
Triple-Sugar Iron (TSI) Agar Test
differentiate between various gram-negative bacilli; 7 ingredients: 1) 0.1% glucose (2) 1.0% sucrose (3) 1.0% lactose (4) peptones (5) phenol red -pH indicator (6) sodium thiosulfate (7) ferrous sulfate (hydrogen sulfide gas indicator); Glucose, sucrose and lactose are carb substrates (fermentation of carbs into acid-end products results in yellow color on slant/or butt as acids react with phenol red. All orgs will preferentially ferment glucose before fermenting lactose and/or sucrose. Gas production determined by observing butt of tube for gas bubbles or cracks in agar; Hydrogen sulfide production- sodium thiosulfate used as substrate, produces H2S, reacts with ferrous sulfate (H2S indicator), causes black precipitate
Interpretation of TSI slants
a. Red (alk) slant and yellow (acid) butt: only glucose fermentation occurred
b. Yellow (acid) slant and yellow (acid) butt: lactose and/or sucrose fermentation occurred
c. Red (alk) slant and red or orange-red (alk) butt: no carb fermentation has occurred; peptones are catabolized resulting in alkaline pH due to production of ammonia
Urease Test
hydrolytic enzyme called urease; breaks down covalent carbon-nitrogen bonds in compound, hydrolyzing urea into ammonia, carbon dioxide and water; urea broth tubes used to determine if urease-positive or negative; urea as substrate and phenol red as pH indicator; urease-positive ammonia produced by urea hydrolysis will raise pH and react with pH indicator, creates “hot pink” color;
Positive = hot pink color
Negative = no color change in media
SIM agar Test
contains amino acid tryptophan, peptones, sodium thiosulfate, and ferrous sulfate; tests for 3 things (1) indole production (2) hydrogen sulfide production (3) motility
Indole production in SIM test
if org produces tryptophanase enzyme, will degrade tryptophan into indole, pyruvate, and ammonia; to test, inoculated and incubated for 24 hours, then solution (Kovac’s Reagent) added to tube. Contains HCl, Butanol, and pdaba; acidified butanol extracts indole and brings it to surface, reacts with pdaba turning a cherry red
Hydrogen Sulfide production in SIM test
orgs which produce thiosulfate reductase can reduce sulfur to hydrogen sulfide gas; source of sulfur is sodium thiosulfate in tubes; H2S gas produced, reacts with ferrous sulfate (black precipitate)
Motility in SIM test
motile orgs will exhibit diffuse growth that spreads out from initial stab line
consists of two separate components: Methyl Red and Vogues Proskauer Test; purpose is to determine if organism is able to ferment glucose and if so, what types of fermentation acids produced. Bacteria initially grown in MRVP broth (contains peptone, glucose, and phosphate buffer)
Methyl Red (MR) Test
initially designed to distinguish between Escherichia coli and Enterobacter aerogenes; after incubation of organism, methyl red is added to tube to determine pH. Red color indicates positive MR reaction indicating pH lower than 4.4 (due to production of fermentation acids)
Vogues-Proskauer Test
purpose is to detect bacteria which ferment glucose, but produce only one acid end-product, usually acetic acid. Acetic acid produced initially lowers pH of media, but is converted to acetylmethylcarbinol (leads to pH of about 6.2). After incubation, Barritt’s Reagent A and B (40% KOH) added to media; chemicals in reagents react with acetylmethcarbinol; Positive reaction = dark red band at top of broth; fermented glucose to produce acetic acid only (an organism will NEVER be positive for both MR and VP tests!
Citrate Utilization Test
determines if organism can use citrate as carbon source; slant contains sodium citrate, bromthymol blue (pH indicator), sodium, and water; if able to utilize citrate, citrase enzyme will break citrate into oxaloacetic acid and acetic acid. Oxaloacetic acid is broken down into pyruvate and acetic acid converted to carbon dioxide. CO2 reacts with water and sodium in media to produce alkaline sodium carbonate; sodium carbonate reacts with pH indicator to produce “Prussian Blue” color (positive result)
Nitrate Reduction Test
used to determine if organisms contain nitrate reductase enzyme which allows reduction of nitrate into nitrite; after incubation, reagents added. Nitrate Reagent A added first, Nitrate Reagent B added next. Red color = positive for nitrate reduction (presence of nitrites)
No color, pinch of zinc added; if red color appears = negative for nitrite reduction (zinc has reduced nitrate to nitrite causing red color)
No color change after zinc added = nitrates reduced beyond nitrites to ammonia or molecular nitrogen, org is positive for nitrate reduction
Nitrate Reagent A for Nitrate Reduction Test
contains sulfanilic acid
Nitrate Reagent B for Nitrate Reduction Test
contains alpha-naphthylamine
Nitrate Broth for Nitrate Reduction test
contains beef extract, peptone, and nitrate
Lysine Iron Agar Test
determines if organism is able to carry out lysine decarboxylation or lysine deamination; slant contains lysine, glucose, peptones
Lysine decarboxylation
anaerobic process; fermentation of glucose- some organisms will synthesize lysine decarboxylase enzyme (cleaves carboxyl group from lysine); creates diamine end-product called cadaverine (raises pH level above 6.8), reacts with pH indicator to give dark purple color in butt of tube
Ferment glucose but unable to synthesize lysine decarboxylase- bacteria will produce acid end products that lower pH (results in yellow color in butt of tube)
Lysine deamination
aerobic reaction; ability to deaminate lysine- ammonia produced will react with ferric ammonium citrate to produce alpha-ketocarboxylic acid (produces dark red color on slant of tube). Requires oxygen (never occurs in butt of tube)
Lysine Iron Agar
slant contains lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate and ferric ammonium citrate
Bromcresol purple is dark purple color at pH 6.8 or higher, turns yellow at pH below 5.2
Results: slant- purple color = negative deamination, red color = positive deamination; butt- purple color = positive decarboxylation, yellow color = negative decarboxylation
Note: red slant, nearly ALWAYS yellow butt
Hydrogen Sulfide production in LIA
tests for hydrogen sulfide production; organisms that produce thiosulfate reductase enzyme can reduce sulfur to hydrogen sulfide gas; source in tubes is sodium thiosulfate. If produced, reacts with ferric ammonium citrate, giving black precipitate
Yogurt production
milk heated to point of boiling (to kill any bacteria present), then cooled so that lactic acid bacteria are added (not destroyed). After milk cooled, two active cultures added: streptococcus thermophilus and lactobacillus bulgaricus; casein (milk protein) exists as colloidal suspension (calcium caseinate)- gives milk white color and turbid appearance, lactic acid produced during lactose fermentation
Streptococcus thermophilus
rapidly ferments lactose in milk to lactic acid; pH in milk decreases;
Lactobacillus bulgaricus
ferments remaining lactose in milk; more acid-tolerant bacterium, can withstand already present lactic acid
Indicator organisms
fecal coliforms; indicates water contaminated with human or animal feces
Presumptive Test
series of tubes of phenol red lactose broth are inoculated with measured amounts of water to see if contains any lactose-fermenting bacteria that produce gas; fermentation plus gas seen in lactose broth, presumed coliforms are in water sample
Most probable number
by counting the number of positive tubes positive for gas production at each dilution, the “most probable number” (MPN) of coliforms is statistically determined using a standardized chart
Confirmed Test
involve plating bacteria from lactose broth tube onto differential media such as EMB or MacConkey to confirm presence of fecal coliforms
Completed test
involve testing for various biochemical properties (MRVP, citrate, urease, indole, etc.) to determine genus and species of coliforms present
Salmonella-Shigella Agar plates
selective and differential; contain lactose, bile salts, ferric citrate, and neutral red; bile salts selective for gram-negative enteric bacteria; lactose carb substrate allows differentiation between fermentors and non-fermenters; if lactose fermentation, acid end-products react with neutral red pH indicator giving bacterial growth pink-red color; ferric citrate acts as indicator of hydrogen sulfide production (black precipitate when H2S present)
Escherichia on SS plates
and other fecal forms such as K. pneumoniae: ferment lactose and produce reddish pink colonies on agar
Salmonella on SS plates
do not ferment lactose; produce hydrogen sulfide; colonies either black or clear with black dot on center
Shigella on SS plates
do not ferment lactose; do not produce hydrogen sulfide; colonies will be clear
Columbia C-CNA agar
selective media which selects for Gram-positive bacteria. It contains two antibiotics, colistin and naladixic acid, which inhibit the growth of gram-negative bacteria, thus selecting for Gram-positive organisms. C-CNA agar also contains whole blood, which allows differentiation based on hemolysis patterns
GasPak jar or Aneropak
creates an oxygen-free environment for the growth of anaerobic microorganisms; anaerobic conditions created by adding water to gas generator envelope placed in jar just before sealing; envelope contains two chemical tablets, sodium borohydride and sodium bicarbonate; Water reacts with chemicals, producing hydrogen gas and carbon dioxide; hydrogen gas combines with free oxygen in chamber to produce water, removing all free oxygen from chamber; catalyzed by palladium, carbon dioxide replaces removed oxygen