You are on page 1of 75

PREPARATION OF SERIAL

DILUTION OF BACTERIAL
SUSPENSION
BY: NELVEN M. GALLEGO, RMT, MLS(ASCPI)
MAAM GINA SADANG, RMT, MSMT

Exercise No. 13

Serial Dilution of
Bacterial Suspension
is a very significant
step in determining
bacterial colony count
NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Exercise No. 13

Culture Media Plates

having 30-300 colonies
are considered for
colony counting

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

5% McFarland standard NELVEN M.Materials ¨  Original bacterial suspension ¨  Sterilized Wassermann test tubes ¨  Test tube rack ¨  Sterilized serological pipette ¨  Sterile distilled H2O ¨  O. RMT. GALLEGO. MLS(ASCPi) .

1:10.000 & 1:100. GALLEGO. 1:100. 1:1. MLS(ASCPi) .•  Sterilize the Wassermann test tubes •  Label the test tubes (1:10.000.5 mL of Distilled H2O on each tube NELVEN M. RMT.000) •  Deliver 4.

GALLEGO. RMT. MLS(ASCPi) .Procedure NELVEN M.

RMT. Prepare a serial dilution by transferring 0. GALLEGO. MLS(ASCPi) .5% McFarland standard.Procedure Using the Original Bacterial Suspension Compare the turbidity with 0.5mL NELVEN M.

Procedure NELVEN M. GALLEGO. MLS(ASCPi) . RMT.

RMT.Procedure Bacterial Suspension Save the diluted bacterial suspensions for future use NELVEN M. MLS(ASCPi) . GALLEGO.

GALLEGO. MLS(ASCPI) MAAM GINA SADANG. MSMT . RMT. RMT.PREPARATION OF SPREAD PLATE FOR COLONY COUNT BY: NELVEN M.

Exercise No. MLS(ASCPi) . RMT. 14 Spread Plate Method Measures the number of viable bacterial cell in a milliliter of culture NELVEN M. GALLEGO.

GALLEGO. MLS(ASCPi) .Exercise No. RMT. 14 NELVEN M.

GALLEGO.Exercise No. RMT. 14 The number of colonies counted (30-300 colonies/plate) Is multiplied by the reciprocal of the dilution to get the number of bacteria for every millimeter of the original bacterial suspension NELVEN M. MLS(ASCPi) .

MLS(ASCPi) . GALLEGO.Exercise No. 14 Plates with colonies <30 is “too few to count” and those with colonies >300 is “too many to count” NELVEN M. RMT.

14 NELVEN M. GALLEGO. MLS(ASCPi) .Exercise No. RMT.

Materials ¨  Serially diluted bacterial suspension ¨  Sterile serological pipette ¨  Trypticase soy agar ¨  Bent glass rod spreaders ¨  Quebec colony counter NELVEN M. MLS(ASCPi) . GALLEGO. RMT.

deliver 0. MLS(ASCPi) .Procedure Using a sterile serological pipette. GALLEGO. RMT.1mL of the serially diluted bacterial suspension (5 Dilutions) to 5 different TSA. NELVEN M.

RMT.Procedure NELVEN M. MLS(ASCPi) . GALLEGO.

Procedure Spread the bacterial suspension on the surface of the agar using a bent glass rod spreader Turn the plate clockwise and repeat the spreading ensuring that the entire surface is “lawned” with the bacterial suspension NELVEN M. GALLEGO. RMT. MLS(ASCPi) .

Procedure Plates are inverted. wrapped. MLS(ASCPi) . GALLEGO. and incubated at 37 centigrade for 18-24 hours NELVEN M. RMT.

count the number of bacterial colonies (30-300) using a Quebec colony counter Compute for the average number of bacterial cell NELVEN M. RMT. MLS(ASCPi) . GALLEGO.Procedure After incubation.

RMT. RMT.Thanks for Listening J ¨  Get ¼ sheet of paper ¨  Prepared by: Nelven M. Gallego. MLS(ASCPi) NELVEN M. GALLEGO. MLS(ASCPi) .

IDENTIFICATION OF GRAM
POSITIVE COCCI
BY: NELVEN M. GALLEGO, RMT, MLS(ASCPI)
MAAM GINA SADANG, RMT, MSMT

Exercise No. 16

Gram positive
microorganisms are normal
inhabitants of human skin &
mucous membranes
Infections caused by these
microorganisms can spread
through direct contact w/
infected person or fomites
NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Exercise No. 16

Medically significant Gram
positive cocci include
Staphylococcus &
Streptococcus
Although both genera contain
large amount of peptidoglycan

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

CAP and PEA using a simple streaking NELVEN M. MLS(ASCPi) . RMT. GALLEGO. Isolation of G+ cocci Day1 Inoculate the bacterial suspension of G+ organism on BAP.

Procedure NELVEN M. GALLEGO. RMT. MLS(ASCPi) .

MLS(ASCPi) . RMT. NaCl. microorganisms BAP Tryptones. agar.Blood Agar Plate Basal medium + Cultivation of enriched substance moderately 5-10% Sheep/rabbit/ fastidious horse/human blood. types of 5% blood: Extracellular enzymes hemolysis NELVEN M. GALLEGO. soybean Differentiation of digest.

CAP 5-10% Sheep. MLS(ASCPi) .Chocolate Agar Plate Basal medium + Cultivation of enriched Haemophilus and substance other fastidious spp. GALLEGO. horse or release Hb and human NAD NELVEN M. RMT. Warm enough to lyse RBC and rabbit.

MLS(ASCPi) . aeruginosa is not inhibited.rods may grow on PEA P.Phenyl Ethyl Alcohol 5% Sheeps blood primarily Phenylethyl alcohol to isolate G+ cocci such as inhibits facultative G- Enterococci. Staph and Strep from specimens with rods. but colonies are than 24H of incubation. especially mixed microbiota swarming Proteus spp PEA G. Some G+ cocci may require more agar. GALLEGO. Although it smaller than usual and contains blood it is not be used in can be readily differentiated the interpretation of hemolytic from those of G+ rods reactions NELVEN M. RMT.

GALLEGO. MLS(ASCPi) . RMT.Swarming of Proteus NELVEN M.

MLS(ASCPi) . RMT. GALLEGO. NELVEN M.Procedure Day1 Incubate BAP and PEA at 37 degrees celcius for 18-24 hours.

RMT.Procedure Day1 CAP is incubated in an anaerobic environment using a candle jar. GALLEGO. NELVEN M. MLS(ASCPi) .

Bacterial Diversity and Umbiquity Capnophiles •  are microorganisms that thrive in the presence of high concentrations of carbon dioxide Obligate anaerobe •  is any organism that does not require oxygen for growth NELVEN M. RMT. GALLEGO. MLS(ASCPi) .

RMT.Anaerobic Cultivation Gas pack Candle Jar NELVEN M. GALLEGO. MLS(ASCPi) .

MLS(ASCPi) . Exercise No. 16 Day2 They can be differentiated by their reaction to different tests such as hemolysis test and catalase test NELVEN M. GALLEGO. RMT.

MLS(ASCPi) . RMT. Procedure Day2 Interpret the result basing on the hemolytic characteristic (BAP and CAP) and colony size (PEA) NELVEN M. GALLEGO.

GALLEGO.Types of Hemolysis NELVEN M. MLS(ASCPi) . RMT.

Types of Hemolysis NELVEN M. GALLEGO. MLS(ASCPi) . RMT.

GALLEGO. MLS(ASCPi) .Types of Hemolysis NELVEN M. RMT.

RMT. GALLEGO.Phenyl Ethyl Alcohol agar NELVEN M. MLS(ASCPi) .

GALLEGO. Pinhead colonies NELVEN M. MLS(ASCPi) . RMT.Pinpoint vs.

Gram stain G+ cocci Catalase NELVEN M. GALLEGO. MLS(ASCPi) . RMT.

RMT. Catalase test Day2 Perform the catalase test by mixing the microorganism w/ 1 drop 3% H2O2 in clean glass slide. Observe for bubbling or effervescence NELVEN M. GALLEGO. MLS(ASCPi) .

RMT. GALLEGO.Catalase test NELVEN M. MLS(ASCPi) .

Catalase test NELVEN M. RMT. GALLEGO. MLS(ASCPi) .

RMT. MLS(ASCPi) . GALLEGO.Catalase test NELVEN M.

GALLEGO. RMT. MLS(ASCPi) .Catalase test Catalase Positive Negative Slide Tube Coagulase Coagulase NELVEN M.

GALLEGO. RMT. MLS(ASCPi) . Observe for clumping indicating presence of enzyme-bound coagulase NELVEN M. Procedure Day2 Proceed to slide coagulase test by transferring the microorganism to a slide containing 1 drop of plasma.

RMT.5mL of human plasma. Incubate at 37 degrees Celsius for 4 hours NELVEN M. MLS(ASCPi) . GALLEGO.Procedure Day2 Perform the tube coagulase test by inoculating on a tube containing 0.

Procedure Day2 Observe clot formation at 30 minute interval. RMT. GALLEGO. MLS(ASCPi) . Formation of solid clot indicates the presence of enzyme free coagulase NELVEN M.

NELVEN M. RMT. MLS(ASCPi) . GALLEGO.

RMT. MLS(ASCPi) . GALLEGO.Procedure Day2 Inoculate the microorganism on MSA. Incubate at 37 degree Celsius for 18-24 hours NELVEN M.

MLS(ASCPi) . MSA S. RMT. aureus can ferment This lowers the pH and mannitol. Selective and (7. the sole changes the color of carbohydrate in the medium. to produce acid products phenol red. to yellow NELVEN M.5% ) inhibits most gram-negative and gram- differential medium positive bacteria except Staphlococcus spp.Mannitol Salt Agar A high salt conc. GALLEGO. the pH indicator.

MLS(ASCPi) . RMT.Procedure NELVEN M. GALLEGO.

Isolation of Enterics Plates Plates Plates EMB HEA SSA Mac XLD BSA NELVEN M. RMT. GALLEGO. MLS(ASCPi) .

G.MacConkey Agar Selective. GALLEGO. Lactose as the sole CHO source. primary produces red or pink plating medium for colonies w/ precipitated enterics bile MacConkey Acid production from LF G+ organisms inhibited by causes the the neutral Crystal violet and bile red dye as an indicator salts NELVEN M. RMT.rods that is LF differential. MLS(ASCPi) .

NELVEN M. GALLEGO. RMT. MLS(ASCPi) .

GALLEGO. RMT. MLS(ASCPi) .MAC MACCONKEY AGAR LACTOSE FERMENTER DARK PINK COLONY LATE-LACTOSE LIGHT PINK COLONY FERMENTER NON-LACTOSE WHITE/ DIRTY WHITE FERMENTER COLONY NELVEN M.

RMT. GALLEGO. MLS(ASCPi) .Eosin Methylene Blue Peptone base Eosin Y & containing lactose methylene blue as indicators and & sucrose selective ingredients EMB Fermentation is detected by color changes and Sucrose serves as an alternative CHO source precipitation of the incorporated dyes as the for slow-lactose fermenters pH drops NELVEN M.

Eosin Methylene Blue NELVEN M. GALLEGO. RMT. MLS(ASCPi) .

MLS(ASCPi) . GALLEGO. RMT.Eosin Methylene Blue EMB EOSIN METHYLENE BLUE AGAR LACTOSE DARK PINK COLONY FERMENTER LATE-LACTOSE LIGHT PINK COLONY FERMENTER NON-LACTOSE WHITE/ COLORLESS FERMENTER COLONY NELVEN M.

GALLEGO.Hektoen Enteric Agar Selective Selective ingredients differential medium are bile salts. ferric also the growth of ammonium citrate): many G.organisms Hydrogen sulfide gas NELVEN M. pH: used for direct isolation Bromthymol blue of enteric pathogen from feces & acid fuchsin HEA Not only inhibit the Ferric salts (Na growth of G+ but thiosulfate. RMT. MLS(ASCPi) .

Hektoen Enteric Agar HEA HEKTOEN ENTERIC AGAR LACTOSE NON-PATHOGENIC FERMENTER ORANGE/ SALMON ORANGE NON. PATHOGENIC/ GREEN OR LACTOSE BLUE COLOR FERMENTER NELVEN M. MLS(ASCPi) . RMT. GALLEGO.

in some formulations.Thiosulfate Bile Salt Sucrose Selective medium TCBS agar is also used to isolate Vibrio differential in that spp. sodium and. from stool specimens Vibrio spp. may produce having mixed biota characteristic colonies TCBS Bromthymol blue Sodium citrate. GALLEGO. normally present in stool NELVEN M. RMT. thiosulfate inhibits G thymol blue are incorporated to indicate +cocci and G-rods the pH. MLS(ASCPi) .

Thiosulfate Bile Salt Sucrose NELVEN M. MLS(ASCPi) . RMT. GALLEGO.

RMT. MLS(ASCPi) . sodium citrate. GALLEGO. which inhibit the carbohydrate source in the growth of G+ and many medium. and brilliant Lactose is the sole green.rods normally is the pH indicator NELVEN M.Salmonella Shigella Agar (SS) agar is used to select SS agar is also for Salmonella and differential in that these organisms produce some strains of Shigella characteristic colonies on from stool specimens. the medium SSA Bile salts. G. and neutral red LF.

it will precipitate in the produce acid and center of the colony change the indicator to pink-red NELVEN M. it is added as a source of sulfur for the production reacts with the ferric of H2S. GALLEGO.Salmonella Shigella Agar Sodium thiosulfate If H2S is produced. MLS(ASCPi) . ammonium citrate SSA If an organism ferments forming a black lactose.RMT..

MLS(ASCPi) .Salmonella Shigella Agar NELVEN M. RMT. GALLEGO.

Xylose Lysine Deoxycholate (XLD) agar is selective The salt. indicator to turn red NELVEN M. inhibits Shigella spp. which results in a pH increased acidity from increase that causes the pH carbohydrate. RMT. MLS(ASCPi) . and many G.rods that are not enteric pathogens and inhibits Salmonella spp G+ organisms XLD A phenol red indicator decarboxylation of in the medium detects lysine. GALLEGO. sodium and differential for desoxycholate.

RMT. GALLEGO.Xylose Lysine Deoxycholate NELVEN M. MLS(ASCPi) .

Xylose Lysine Desoxycholate NELVEN M. MLS(ASCPi) . GALLEGO. RMT.

RMT. which inhibit the growth of G+.Bismuth Sulfite Agar The selective ingredients are Selective medium bismuth sulfite and brilliant for the isolation of green. insoluble to produce ferric sufide precipitate NELVEN M. microbiota. GALLEGO. and Shigella BSA the ferrous sulfate in which is deposited in the this medium is reactive bacterial colony as a with hydrogen sulfide black. MLS(ASCPi) . most LF intestinal normal Salmonella spp.

RMT. GALLEGO. MLS(ASCPi) .Bismuth Sulfite Agar NELVEN M.

MLS(ASCPi) NELVEN M. GALLEGO. Gallego. MLS(ASCPi) .Thanks for Listening J ¨  Prepare for a 30 items Quiz on Wednesday nextweek ¨  Prepared by: Nelven M. RMT. RMT.