Micropara 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)
Exercise No. 13
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
Exercise No. 14
Spread Plate Method
Measures the number

of viable bacterial cell
in a milliliter of culture

Exercise No. 14

Exercise No. 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

Exercise No. 14
Plates with colonies <30

is too few to count and
those with colonies >300
is too many to count

Exercise No. 14

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 J
Get sheet of paper
Prepared by: Nelven M. Gallego, RMT, MLS(ASCPi)

IDENTIFICATION OF GRAM
POSITIVE COCCI
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
Exercise No. 16
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 + Cultivation of

enriched substance moderately
5-10% Sheep/rabbit/ fastidious
horse/human blood. microorganisms
BAP
Tryptones, soybean Differentiation of
digest, NaCl, agar, types of
5% blood:
Extracellular enzymes hemolysis
Chocolate Agar Plate
Basal medium + Cultivation of

enriched Haemophilus and
substance other fastidious spp.
CAP
5-10% Sheep, Warm enough to
lyse RBC and
rabbit, horse or release Hb and
human NAD
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, especially
mixed microbiota swarming Proteus spp
PEA
G- rods may grow on PEA P. aeruginosa is not inhibited.
Some G+ cocci may require more
agar, but colonies are
than 24H of incubation. 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
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

Exercise No. 16
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

Types of Hemolysis

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 Tube
Coagulase 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
A high salt conc.

Selective and (7.5% ) inhibits most
gram-negative and gram-
differential medium positive bacteria except
Staphlococcus spp.
MSA
S. aureus can ferment This lowers the pH and
mannitol, the sole changes the color of
carbohydrate in the medium, the pH indicator,
to produce acid products phenol red, to yellow
Procedure

Isolation of Enterics
Plates Plates Plates

EMB HEA SSA
Mac XLD BSA

MacConkey Agar
Selective, Lactose as the sole CHO
source. G- rods that is LF
differential, 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
MAC MACCONKEY AGAR
LACTOSE FERMENTER DARK PINK COLONY
LATE-LACTOSE LIGHT PINK COLONY

FERMENTER
NON-LACTOSE WHITE/ DIRTY WHITE

FERMENTER COLONY

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

EMB EOSIN METHYLENE BLUE

AGAR
LACTOSE DARK PINK COLONY
FERMENTER
LATE-LACTOSE LIGHT PINK COLONY
FERMENTER
NON-LACTOSE WHITE/ COLORLESS

FERMENTER COLONY

Hektoen Enteric Agar
Selective Selective ingredients
differential medium are bile salts. 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, ferric
also the growth of ammonium citrate):
many G- organisms Hydrogen sulfide gas
Hektoen Enteric Agar
HEA HEKTOEN ENTERIC AGAR
LACTOSE NON-PATHOGENIC
FERMENTER ORANGE/ SALMON
ORANGE
NON- PATHOGENIC/ GREEN OR
LACTOSE BLUE COLOR
FERMENTER
Thiosulfate Bile Salt Sucrose
Selective medium TCBS agar is also

used to isolate Vibrio differential in that
spp. from stool specimens Vibrio spp. may produce
having mixed biota characteristic colonies
TCBS
Bromthymol blue Sodium citrate, sodium
and, in some formulations, thiosulfate inhibits G
thymol blue are
incorporated to indicate +cocci and G-rods
the pH. normally present in stool
Thiosulfate Bile Salt Sucrose

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, sodium
citrate, and brilliant Lactose is the sole
green, which inhibit the carbohydrate source in the
growth of G+ and many medium, and neutral red
LF, G- rods normally is the pH indicator
Sodium thiosulfate If H2S is produced, it

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

Xylose Lysine Deoxycholate
(XLD) agar is selective The salt, sodium
and differential for desoxycholate, inhibits
Shigella spp. 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, which results in a pH
increased acidity from increase that causes the pH
carbohydrate. indicator to turn red
Xylose Lysine Deoxycholate

Xylose Lysine Desoxycholate

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

Thanks for Listening J
Prepare for a 30 items Quiz on

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

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PREPARATION OF SERIAL

Culture Media Plates

having 30-300 colonies

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Original bacterial suspension

Test tube rack

Sterilized serological pipette

Sterile distilled H2O

O.5% McFarland standard

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Label the test tubes (1:10, 1:100,

Deliver 4.5 mL of Distilled H2O on

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Using the Original Bacterial

Compare the turbidity with

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Save the diluted

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Spread Plate Method

Measures the number

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

The number of colonies counted

Is multiplied by the reciprocal of

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Plates with colonies <30

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Serially diluted bacterial

Trypticase soy agar

Bent glass rod spreaders

Quebec colony counter

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Using a sterile serological

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Spread the bacterial suspension

Turn the plate clockwise and

Plates are inverted,

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

After incubation, count the

Compute for the average

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Medically significant Gram

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Day1 Inoculate the bacterial

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Basal medium + Cultivation of

Basal medium + Cultivation of

5% Sheeps blood primarily Phenylethyl alcohol

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Day1 Incubate BAP and PEA

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Gas pack Candle Jar

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Day2 They can be differentiated by

NELVEN M. GALLEGO, RMT, MLS(ASCPi)

Day2 Interpret the result basing on the

NELVEN M. GALLEGO, RMT, MLS(ASCPi)