Nelvens-Final-LabNotes

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)


Culture Media Plates 

having 30-300 

colonies are considered 

for colony counting 


Materials 

Original bacterial suspension 

Sterilized Wassermann test tubes 

Test tube rack 

Sterilized serological pipette 

Sterile distilled H2O 

O.5% McFarland standard 


• Sterilize the Wassermann test 

tubes 

• Label the test tubes (1:10, 1:100, 

1:1,000, 1:10,000 & 1:100,000) 

• Deliver 4.5 mL of Distilled H2O on 

each tube 


Procedure 


Procedure 

Using the Original Bacterial 

Suspension 

Compare the turbidity with 

0.5% McFarland standard. 

Prepare a serial dilution by 

transferring 0.5mL 


Procedure 


Procedure 

Bacterial Suspension 

Save the diluted 

bacterial suspensions 

for future use 


PREPARATION OF SPREAD PLATE 

FOR COLONY COUNT 




Spread Plate Method 

Measures the number 

of viable bacterial cell 

in a milliliter of culture 





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 



Plates with colonies 300 

is “too many to count” 




Materials 

Serially diluted bacterial 

suspension 

Sterile serological pipette 

Trypticase soy agar 

Bent glass rod spreaders 

Quebec colony counter 


Procedure 

Using a sterile serological 

pipette, deliver 0.1mL of 

the serially diluted 

bacterial suspension (5 

Dilutions) to 5 different TSA. 


Procedure 


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 


Procedure 

Plates are inverted, 

wrapped, and 

incubated at 37 

centigrade for 18- 

24 hours 


Procedure 

After incubation, count 

the number of bacterial 

colonies (30-300) using a 

Quebec colony counter 

Compute for the average 

number of bacterial cell 


Thanks for Listening 

Get ¼ sheet of paper 

Prepared by: Nelven M. Gallego, RMT, MLS(ASCPi) 


IDENTIFICATION OF GRAM 

POSITIVE COCCI 




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 



Medically significant Gram 

positive cocci include 

Staphylococcus & 

Streptococcus 

Although both genera 

contain large amount of 

peptidoglycan 


Isolation of G+ cocci 

Day1 Inoculate the bacterial 

suspension of G+ organism on 

BAP, CAP and PEA using a 

simple streaking 


Procedure 


Blood Agar Plate 

Basal medium + 

enriched substance 5- 

10% 

Sheep/rabbit/horse/h 

uman blood. 

Cultivation of 

moderately 

fastidious 

microorganisms 

BAP 

Tryptones, soybean 

digest, NaCl, agar, 

5% blood: 

Extracellular enzymes 

Differentiation of 

types of 

hemolysis 


Chocolate Agar Plate 

Basal medium + 

enriched 

substance 

Cultivation of 

Haemophilus and 

other fastidious spp. 

CAP 

5-10% Sheep, 

rabbit, horse or 

human 

Warm enough to 

lyse RBC and 

release Hb and 

NAD 


Phenyl Ethyl Alcohol 

5% Sheeps blood primarily 

to isolate G+ cocci such as 

Enterococci, Staph and 

Strep from specimens with 

mixed microbiota 

Phenylethyl alcohol 

inhibits facultative G- 

rods, especially 

swarming Proteus spp 

PEA 

G- rods may grow on PEA 

agar, but colonies are 

smaller than usual and 

can be readily differentiated 

from those of G+ rods 

P. aeruginosa is not inhibited. 

Some G+ cocci may require more 

than 24H of incubation. Although it 

contains blood it is not be used in 

the interpretation of hemolytic 

reactions 


Swarming of Proteus 


Procedure 

Day1 

Incubate BAP and PEA 

at 37 degrees celcius 

for 18-24 hours. 


Procedure 

Day1 

CAP is incubated in an 

anaerobic environment 

using a candle jar. 


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 


Anaerobic Cultivation 

Gas pack 

Candle Jar 



Day2 They can be differentiated by 

their reaction to different tests 

such as hemolysis test and 

catalase test 


Procedure 

Day2 Interpret the result basing on the 

hemolytic characteristic 

(BAP and CAP) and colony 

size (PEA) 


Types of Hemolysis 






Phenyl Ethyl Alcohol agar 


Pinpoint vs. Pinhead colonies 


Gram stain 

G+ cocci 

Catalase 


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 


Catalase test 


Catalase test 


Catalase test 


Catalase test 

Catalase 

Positive 

Negative 

Slide 

Coagulase 

Tube 

Coagulase 


Procedure 

Day2 Proceed to slide coagulase test 

by transferring the microorganism to a 

slide containing 1 drop of plasma. 

Observe for clumping indicating 

presence of enzyme-bound 

coagulase 


Procedure 

Day2 

Perform the tube 

coagulase test by 

inoculating on a tube 

containing 0.5mL of human 

plasma. Incubate at 37 

degrees Celsius for 4 hours 


Procedure 

Day2 Observe clot formation 

at 30 minute interval. 

Formation of solid clot 

indicates the presence of 

enzyme free 

coagulase 



Procedure 

Day2 

Inoculate the 

microorganism on 

MSA. Incubate at 37 

degree Celsius for 

18-24 hours 


Mannitol Salt Agar 

Selective and 

differential medium 

A high salt conc. (7.5% 

) inhibits most gramnegative 

and grampositive 

bacteria except 

Staphlococcus spp. 

MSA 

S. aureus can ferment 

mannitol, the sole 

carbohydrate in the medium, 

to produce acid products 

This lowers the pH and 

changes the color of 

the pH indicator, 

phenol red, to yellow 


Procedure 


Isolation of Enterics 

Plates 

Plates 

Plates 

EMB 

HEA 

SSA 

Mac 

XLD 

BSA 


MacConkey Agar 

Selective, 

differential, primary 

plating medium for 

enterics 

Lactose as the sole CHO 

source. G- rods that is LF 

produces red or pink 

colonies w/ precipitated 

bile 

MacConkey 

Acid production from LF 

causes the the neutral 

red dye as an indicator 

G+ organisms inhibited by 

Crystal violet and bile 

salts 



MAC 

MACCONKEY AGAR 

LACTOSE FERMENTER DARK PINK COLONY 

LATE-LACTOSE 

FERMENTER 

NON-LACTOSE 

FERMENTER 

LIGHT PINK COLONY 

WHITE/ DIRTY WHITE 

COLONY 


Eosin Methylene Blue 

Peptone base 

containing lactose 

& sucrose 

Eosin Y & 

methylene blue as 

indicators and 

selective ingredients 

EMB 

Fermentation is detected 

by color changes and 

precipitation of the 

incorporated dyes as the 

pH drops 

Sucrose serves as an 

alternative CHO source 

for slow-lactose 

fermenters 





EMB 

LACTOSE 

FERMENTER 

LATE-LACTOSE 

FERMENTER 

NON-LACTOSE 

FERMENTER 

EOSIN METHYLENE BLUE 

AGAR 

DARK PINK COLONY 

LIGHT PINK COLONY 

WHITE/ COLORLESS 

COLONY 


Hektoen Enteric Agar 

Selective 

differential medium 

used for direct isolation 

of enteric pathogen from 

feces 

Selective ingredients 

are bile salts. pH: 

Bromthymol blue 

& acid fuchsin 

HEA 

Not only inhibit the 

growth of G+ but 

also the growth of 

many G- organisms 

Ferric salts (Na 

thiosulfate, ferric 

ammonium citrate): 

Hydrogen sulfide gas 


Hektoen Enteric Agar 

HEA 

LACTOSE 

FERMENTER 

NON- 

LACTOSE 

FERMENTER 

HEKTOEN ENTERIC AGAR 

NON-PATHOGENIC 

ORANGE/ SALMON 

ORANGE 

PATHOGENIC/ GREEN OR 

BLUE COLOR 


Thiosulfate Bile Salt Sucrose 

Selective medium 

used to isolate Vibrio 

spp. from stool specimens 

having mixed biota 

TCBS agar is also 

differential in that 

Vibrio spp. may produce 

characteristic colonies 

TCBS 

Bromthymol blue 

and, in some formulations, 

thymol blue are 

incorporated to indicate 

the pH. 

Sodium citrate, sodium 

thiosulfate inhibits 

G+cocci and G-rods 

normally present in stool 


Thiosulfate Bile Salt Sucrose 


Salmonella Shigella Agar 

(SS) agar is used to select 

for Salmonella and 

some strains of Shigella 

from stool specimens. 

SS agar is also 

differential in that 

these organisms produce 

characteristic colonies on 

the medium 

Bile salts, sodium 

citrate, and brilliant 

green, which inhibit the 

growth of G+ and many 

LF, G- rods normally 

found in stool 

SSA 

Lactose is the sole 

carbohydrate source in the 

medium, and neutral red 

is the pH indicator 



Sodium thiosulfate 

is added as a source of 

sulfur for the production 

of H2S. 

If H2S is produced, it 

reacts with the ferric 

ammonium citrate 

SSA 

forming a black 

precipitate in the 

center of the colony 

If an organism ferments 

lactose, it will 

produce acid and 

change the indicator to 

pink-red. 




Xylose Lysine Deoxycholate 

(XLD) agar is selective 

and differential for 

Shigella spp. and 

Salmonella spp 

The salt, sodium 

desoxycholate, inhibits 

many G- rods that are not 

enteric pathogens and inhibits 

G+ organisms 

XLD 

A phenol red indicator 

in the medium detects 

increased acidity from 

carbohydrate. 

decarboxylation of 

lysine, which results in a pH 

increase that causes the pH 

indicator to turn red 


Xylose Lysine Deoxycholate 


Xylose Lysine Desoxycholate 


Bismuth Sulfite Agar 

Selective medium 

for the isolation of 

Salmonella spp. 

The selective ingredients are 

bismuth sulfite and brilliant 

green, which inhibit the growth of 

G+, most LF intestinal normal 

microbiota, and Shigella 

BSA 

the ferrous sulfate in 

this medium is reactive 

with hydrogen sulfide 

to produce ferric sufide 

which is deposited in the 

bacterial colony as a 

black, insoluble 

precipitate 


Bismuth Sulfite Agar 



Prepare for a 30 items Quiz on 

Wednesday nextweek 



BIOCHEMICAL TEST FOR 

ENTERICS 



Triple Sugar Iron 



TSI 

Differentiates glucose 

fermenters from non–glucose 

fermenters; also contains tests 

for sucrose and/or lactose 

fermentation, as well as gas 

production during glucose 

fermentation and H2S 

production. 



Glucose, Maltose, 

Sucrose 

Phenol red is the 

pH indicator. Turns to 

yellow when sugars 

are fermented. 

Sodium 

thiosulfate plus 

Ferric ammonium 

sulfate as H2S 

indicator 

TSI 

Gas bubbles : 

Production of gas 





Lysine Iron Agar 



LIA Measures three parameters 

that are useful for 

identifying 

Enterobacteriaceae (lysine 

decarboxylation, lysine 

deamination, and H2S 

production) 



Contains lysine, glucose, 

and protein, 

bromocresol purple 

(pH indicator) 

Sodium 

thiosulfate/ferric 

Ammonium citrate. 

Purple denotes alkaline 

(K), red color (R), acid (A). 

LIA 

K/K: Organism 

decarboxylates but 

cannot deaminate, 

ferments glucose 

K/A: Organism fermented 

glucose but was unable to 

deaminate or 

decarboxylate lysine 



R/A: Organism 

deaminated lysine 

but could not 

decarboxylate it. 

The lysine deamination 

combines with the ferric 

ammonium citrate, 

forming a burgundy color. 

LIA 

Blackening of the butt 

indicates production of 

H2S. The medium has an 

aerobic slant and an 

anaerobic butt. 

When glucose is 

fermented, the butt of the 

medium becomes acidic 

(yellow) 




Simmons’ Citrate 



Citrate 

Detect organisms capable 

of citrate utilization. Citrate 

as the sole carbon 

source, ammonium salt as 

nitrate. Ammonium salt 

alteration changes pH to 

alkaline, bromthymol blue 

shifts from green to blue. 



Determines whether an 

organism can use 

sodium citrate as a 

sole carbon source. 

Contains ammonium 

salts as the sole 

nitrogen source. 

Bacteria able to 

use citrate will use 

the ammonium salts, 

, releasing 

ammonia 

Citrate 

The alkaline pH that 

results from use of the 

ammonium salts changes 

the pH indicator, 

bromthymol blue 


Christensen’s Urease 



Urease 

Identification of 

Enterobacteriaceae 

species capable of 

producing urease. 

(Citrobacter, Klebsiella, 

Proteus, Providencia, and 

Yersinia spp.) 



Determines whether a 

microorganism can 

hydrolyze urea to 

form ammonia, water, and 

CO2 

Releasing a sufficient 

amount of ammonia to 

produce a color change by 

a pH indicator 

Urease 

The medium contains 

phenol red as the 

pH indicator. 

The resulting alkaline pH 

from hydrolysis of urea is 

indicated by a bright 

pink color 


Sulfide Indole Motility 



SIM is a semisolid agar 

helpful in differentiating 

G- bacteria in the 

Enterobacteriaceae 

An inoculating needle 

is used to make a straight 

stab down the center of 

the medium 

SIM 

The production of H2S 

is indicated by a 

black precipitate 

pink to red color after 

the addition of Kovac’s 

reagent is positive for 

indole 




Methyl Red Vogues Proskauer 


Methyl Red Vogues Proskauer 



Oxidase test 





Prepare for a 25 items Quiz on Friday

Nelvens-Final-LabNotes

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?