Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Laboratory Methods ID of pathogenic bacteria using series of differential tests  differential & selective media  morphology & staining  biochemical properties  antibiotic resistance or susceptibility  plasmid, ribosomal or DNA analysis

Gram Positive Cocci Staphylococci vs Streptococci –Staph aureus  catalase +, coagulase +, &  -hemolytic –Strep  , , or non-hemolytic, catalase -

Catalase Enzyme that converts H 2 O 2  H 2 O and O 2  Visible bubbles when drop of 3% H 2 O 2 is added to culture of catalase + bacteria

Catalse +Catalse -

Catalase - Catalase +

Between Staph Species S aureus  coagulase + (clots plasma) S epidermidis & saprophyticus  coagulase neg –S saprophyticus  novobiocin resistant –S epidermidis  novobiocin sensitive

Lower tube shows fibrin clot following addition of plasma to culture of Stap aureus. Top tube is negative indicating non-pathogenic Staph.

Left: novobiocin resistant Staph saprophyticus Right: novobiocin senstitive Staph epidermidis

Between Strep Species Hemolysis: –Strep pyogenes & agalactiae   hemolytic –Enterococcus  variable hemolysis –Strep viridans & pneumoniae   hemolytic

Beta-hemolysis with clearing of blood agar

Green discloloration of alpha-hemolysis

No hemolysis on blood agar

More Strep Tests Strep pyogenes  bacitracin sensitive Strep agalactiae & Enterococcus  bacitracin resistant Strep viridans  resistant to optochin Strep pneumoniae  sensitive to optochin

Zone of inhibition around optochin disk - presumptive identification of Strep pneumoniae

Motility Tests Many bacteria show motility in media Agar tube inoculated with needle stab Media contains tetrazolium  bacteria reduces to formazan (red color) Location of bacteria determined by location of red color Useful for Borrelia, Vibrio, Salmonella, E coli, etc.

2,3,5-Triphenyltetrazolium chloride (TTC)Formazan (red color)

Left tube: motileCenter: nonmotileRight: control

Oxidase Tests Presence of cytochrome oxidase Neisseria & Pseudomonas + Enterobacteriaceae  neg Test converts colorless tetramethyl-p- phenylenediamine to oxidized form (deep purple/blue)

Oxidase + colony at 12 o’clock, 3 & 9 o’clock cultures are negative

Oxidase test done on paper saturated with oxidase reagent. Pseudomonas on left (+), Staph aureus on right (-).

Enterobacteriaceae API 20 E   series of substrates and indicators that are inoculated with unknown and media Tests for enzymes and carbohydrate utilization Pattern of + and - results compared to a table of known results to identify unknown

Oxidation-Fermentation Tests Mostly used to distinguish fermentative Enterobacteriaceae from oxidative Pseudomonas & Bordetella Grow isolate in low agar content media in presence of different sugar substrates and pH indicator Stab media: one tube sealed with mineral oil to promote anaerobic growth/ other tube unsealed Indicator yellow at pH 6.0/ green at pH 7.1, blue at pH 7.6

Mineral oil covers media in tubes 1 & 3. Pseudomonas in tubes 1 & 2, Shigella in tubes 3 & 4.

Enterobacteriaceae Enterotube  II  12 compartments of various media allows determination of 15 different characteristics of organism Positive results given a number, added in groups and sums combined to create unique 5-digit identifier in computer databank

Question #1 1.The laboratory obtains a surgical wound swab from a 56 year old male four days after undergoing a colectomy for colon cancer. The isolate is gram +, catalase +, coagulase + and  hemolytic. It is most likely a.Streptococcus pyogenes b.Enterococcus faecalis c.Staphylococcus aureus d.Staphylococcus saphrophyticus e.Streptococcus viridans

Question #2 2.The initial data necessary to adequately identify a bacterial unknown are its a.Ribosomal sequence b.Antibiotic resistance pattern c.Motility d.Ability to ferment glucose e.Morphology and gram staining

Question #3 3.Streptococcus viridans and pneumoniae are both  hemolytic, catalase -, gram + cocci. Which test may readily distinguish them? a.Enterotube  II b.Novobiocin resistance c.Motility test d.Oxidase test e.Resistance to optochin