MICRO 1 LABORATORY

EXERCISE 1: BACTERIAL MORPHOLOGY AND TYPES

BASIC FORMS SPECIFIC MORPHOLOGY REPRESENTATIVE BACTERIUM

COCCI 1. Staphylococcus Staphylococcus aureus
2. Streptococci Streptococcus pyogenes
(0.4-2 um) 3. Diplococci Streptococcus pneumonia
Neisseria gonorrhoeae
4. Tetrad Gaffkya tetragena
5. Sarcina Sarcina lutea

BACILLI 1. Streptobacilli Bacillus subtilis

2. Diplobacilli (Snapping & Slipping) Mycobacterium tuberculosis
(0.2-4 um x 0.5-20 um WxL) 3. Coccobacilli Escherichia coli
4. Vibrio Vibrio cholera

SPIRALS 1. Spirillum Campylobacter jejuni

(1-14 um length) 2. Spirochete Borella sp.
Treponema pallidum

Staining METHOD Staining REACTION

1. Gram Stain = 2 groups a. Gram (+) = Purple or Violet
b. Gram (-) = Red
2. Acid Fast Stain = 2 groups a. Acid-fast = Red
b. Non-Acid fast = Blue
3. Dilute Carbol Fuchsin Red or Reddish Brown
4. Fontana Tribondeau Dark Brown or Black with Yellowish Background

Morphology: COCCI
Specific Morphology: Staphylococci
Rep. Bact.: Staphylococcus aureus
Staining Method: Gram Stain
Staining Reaction: Gram (+)

Gram (+) Staphylococci

cocci in grapelike clusters or cocci in irregular clusters
aureus because it is related to the ability of the organism to produce a
golden yellow pigment

Specific Morphology: Streptococci

Rep. Bact.: Streptococcus pyogenes
Staining Method: Gram Stain
Staining Reaction: Gram (+)

Gram (+) Streptococci

cocci in chains in which organisms look like beads on a string
pyogenic = pus-producing
agent of bacterial tonsillitis

Specific Morphology: Diplococci

Rep. Bact.: Streptococcus pneumoniae
Staining Method: Gram Stain
Staining Reaction: Gram (+)

Gram (+) Diplococci

Cocci in pairs - two round cells facing each other
Lanceolate appearance – the shorter ends are facing each other
Source of Smear: SPUTUM
Red cells – Pus cells

unofficial notes rawr merixxaaa


Not included in Ex 1 drawings
but was part of the questioning
unofficial notes rawr
Other Rept. Bact.: Neisseria gonorrhoeae
Coffee-bean shaped – the longer sides of the cells are facing each
other; adjacent sides are flattened
Gram (-) Diplococci
Specific Morphology: Tetrad
Rep. Bact.: Gaffkya tetragena
Staining Method: Gram Stain
Staining Reaction: Gram (+)
Gram (+) Tetrad
Cocci in groups of four (4) cells
Specific Morphology: Sarcina
Rep. Bact.: Sarcina lutea
Staining Method: Gram Stain
Staining Reaction: Gram (+)
Gram (+) Sarcina
Cocci in cubical packets of eight cells
under the microscope if you use an ordinary stain, basically you will
be looking at four cells in front and the other four behind looking like
shadows: it would make the cells appear thick
Morphology: BACILLI
Specific Morphology: Streptobacilli
Rep. Bact.: Bacillus subtilis
Staining Method: Gram Stain
Staining Reaction: Gram (+)
Gram (+) Streptobacilli
bacilli in chains, one rod-shaped organism after the other
long and plump
unstained area in the cell = spores
Bacillus subtilis is a spore-former
Specific Morphology: Diplobacilli
Rep. Bact.: Mycobacterium tuberculosis
Staining Method: Acid Fast Stain
Staining Reaction: Acid Fast
acid fast snapping diplobacilli or acid fast slipping diplobacilli
bacilli in pairs: usually have two bacillus, one after the other
the cells of TB bacilli are long and slender, and some cells may show
metachromatic granules which are seen as dark dots along the body
of the cell
Blue bg / cells – pus cells & epithelial cells
Source: SPUTUM of a tb px
SPECIAL ARRANGEMENTS:
o Snapping diplobacilli: V or inverted V
o Slipping diplobacilli: two bacillus arranged parallel side by
side
merixxaaa
 Specific Morphology: Coccobacilli
Rep. Bact.: Escherichia coli
Staining Method: Gram Stain
Staining Reaction: Gram (-)

Gram (-) Coccobacilli

Short, plump, oval bacilli
Escherichia coli causes Traveller’s Diarrhea

Specific Morphology: Vibrio

Rep. Bact.: Vibrio cholerae
Staining Method: Gram Stain
Staining Reaction: Gram (-)

Gram (-) Vibrio

Comma-shaped bacilli
Best observed at the thin, end portion of the smear because in those
areas the vibrio has the space to curve. In crowded areas, they will
appear as coccobacilli.

Morphology: SPIRALS
Specific Morphology: Spirillum
Rep. Bact.: Campylobacter jejuni
Staining Method: Gram Stain
Staining Reaction: Gram (-)

Gram (-) Spirillum

Appear as “bird in flight” “n” “s” “w”
Spiral organisms whose long axis remains rigid when in motion

Specific Morphology: Spirochete

Rep. Bact.: Borrelia sp.
Staining Method: Dilute Carbol Fuchsin
Staining Reaction:

a spiral organism whose long axis bends when in motion

appearance of an extremely long, undulating bacillary form
Red spiral bacterium with few looser coils
Red cells: smear from BLOOD CULTURE
Borrelia burgdorferi: agent of Lyme disease

Specific Morphology: Spirochete

Rep. Bact.: Treponema pallidum
Staining Method: Fontana Tripondeau
Staining Reaction:

a spiral organism whose long axis bends when in motion

appearance of an extremely long, undulating bacillary form
Slender spiral bacterium with several tight coils
Corkscrew appearance
Brown or black with yellow/yellow orange background
Syphilis; URETHRAL DISCHARGE

Also not included sa lab Specific Morphology: Spirochete

But apilon lang nako c: Rep. Bact.: Leptospira interrogans
unofficial notes rawr merixxaaa
 under the microscope, it looks like a question mark (hence, the
species name)
less tightly coiled which are hook-like bends on the ends of the cell

GRAM POSITIVE (+) GRAM NEGATIVE (-)

Staphylococcus aureus Neisseria gonorrhoeae
Streptococcus pyogenes Escherichia coli
Streptococcus pneumonia Vibrio cholerae
Gaffkya tetragena Campylobacter jejuni
Bacillus subtilis
ACID FAST: Mycobacterium tuberculosis
DILUTE CARBOL FUCHSIN: Borrelia sp.
FONTANA TRIPONDEAU: Treponema pallidum

unofficial notes rawr merixxaaa

 MICRO 1: LABORATORY Mycobacterium tuberculosis
EXERCISE 02: BACTERIAL CELL AND SPECIAL STRUCTURE stain: acid-fast stain
Learning Outcomes:
▪ Enumerate, describe, and understand the special structures found in
the bacterial cells and its functions and composition.
▪ Examine schematic drawings of a bacterial cell, bacteria with flagella
and bacterial spore as classified to shape and position in the bacterial
cell.
▪ Draw and label the special structures from a set of prepared slides of
bacteria focused under OIO.

SET A: SCHEMATIC DRAWINGS 3. Drawings of Spores as Classified According to Shape and Position in the
Draw and label, do not apply any color. Bacterial Cell
1. Bacterial Cell can form within different areas of the vegetative cell
▪ capsule ▪ flagellum bacterial spores
has a highly refractile oval body from within the cell
▪ cell wall ▪ inclusion (large) located at the end of the cell
▪ cytoplasmic membrane ▪ inclusion (small) the stain is on one side 2. Flagella -

protein : Hagelin ; Hlauch ) antigen

▪ cytoplasm with ribosomes ▪ periplasmic space terminal spores
may also appear like a drumstick or a tennis rack Proteus vulgaris (peritrichous, hence, perimotile)
▪ pilus ▪ chromosome stain: gray method
e.g. Clostridium tetani
▪ plasmid
located at the middle of the cell
central spores equal amount of stained cytoplasm on both sides
e.g. Bacillus subtilis
subterminal located between the middle and the end of the cell
spores e.g. Bacillus subtilis

3. Bacterial Spore reason why a bacteria is resistant to heat

Kindly identify and draw which bacteria have the terminal, central, and
subterminal endospore.
Bacillus subtilis subterminal & / or
-

2. Illustrations of Bacteria with Flagella According to Messea’s Classification stain: Gram’s stain central spore
of Bacteria
are fine, filamentous appendages that have whip-form
length in the end
flagella
known to originate from the cell membrane B. Draw the Special Structures Possessed by these Bacteria.
composed of a protein called flagellin Do a detailed drawing for these bacteria. Emphasize the structures. Do not
has a single, polar flagellum forget to label.
monotrichous
e.g. Vibrio cholerae C. jejuni , P aeruginosa
,
.

1. Metachromatic granules made up of metaphosphate

-

has flagellum at each pole seen as darkly stained round bodies found in the cytoplasm of the bacterial
amphitrichous
e.g. Pseudomonas spp. cell or along the body of the organism
lophotrichous Corynebacterium diptheriae
several flagella at one side
stain: Loeffler’s Methylene Blue Clostridium tetani > terminal spore
the body of the bacteria is surrounded by flagella
peritrichous stain: Gram’s stain
e.g. Proteus vulgaris Salmonella typhi
,

no flagella at all
atrichous
considered as non-motile
Shigella dysenteric ,
Klebsiella pneumoniae

AIRAH M.
4. Slime Layer and Caspule
Streptococcus pneumoniae
stain: Anthony stain

Sarcina lutea
stain: Dilute Carbol Fuchsin or India Ink

slimy layer
>

AIRAH M.
 MICRO1 LAB
Course Microbiology 1

Dates @September 7, 2021

Notes Ex.2 – Bacterial Cell & Special Structures

Status

Task Laboratory

Weighting

EX2: Bacterial Cell and Special structures

Objectives:

Identify and draw the parts of a bacterial cell

To recognize and describe the morphologic structures of a bacillus

To identify, differentiate and draw the different special structures of a bacillus and its
composition

A. Schematic Drawings

1. Bacterial Cell

a. electromicrograph of bacillus showing cytoplasm, nuucleus, etc

b. Follow ms vinarta's drawing

2. Drawings of bacteria with flagella according to Messea's classification of

bacteria

MICRO1 LAB 1
 a. Flagella protein : Hagelin ; Hcauch) antigen

is a fine filamentous appendages of uniform lenght and diameter usually

seen in a rod shaped bacteria
> usually ! !!
make the bacteria motile

originated from the cell membrane

take not of composition and significance

i. Monotrichous

Singel polar flagellum

Vibrio cholerae, Campylobacter jejuni, and Pseudomonas

aeruginosa

ii. Lophotrichous

several flagella at one end of cell

Pseudomonas spp

iii. Peritrichous

flagella is found all over the body of the bacteria

Salmonella typhi and Proteus vulgaris

iv. Amphitrichous

single flagellum of each pole of bacteria

Presence of several flagella at both ends

Please draw 2 bacteria

Pseudomonas spp.

v. Atrichous

no flagella present

Klebsiella pneumoniae and Shigella dysenteriae

3. Draiwng of spores as classified according to shape and position in the

bacterial cell

MICRO1 LAB 2
 bacteria resistant to heat
a. Spores
are
-

reason why

seen as highly refractile, oval body or unstained area found

within the cell
& i. Central = equal amount of stain in the cytoplasm

:
ii. Subterminal = unequal amount of stain not in the middle and not
towards the end somewhere 3/4 of the bacterial cell

iii. Terminal = at the end of the bacterial cell; drumstick like

appearance

B. Examine a set of prepared slides of bacteria under OIO and draw the special
structures possessed by these bacteria

1. Metachromatic granules

made up of metaphosphates that is for food reserves or storage granules

a. Corynebacterium diptheriae stained with Loeffler's methylene blue

there are found the cytoplasm of cell and this are seen as darkly stained round
bodies found in the cytoplasm of the bacterial cell or along the body of organism

b. Mycobacterium tuberculosis stained with acid fast stain

MICRO1 LAB 3
 are granular inclusion bodies; also called as volutin granules and they are
present in some bacteria such as the Genus Corynebacterium and Genus
Mycobacterium!!!

are accumulations of metaphosphates formed with the aid of energy yielding

enzymatic reaction
☐

8
volutin granules or bebes.

ernst granules

2. Flagella

a. Proteus vulgaris stained with Gray method

MICRO1 LAB 4
 difficult to visualize that is whhy we have to use special stain which is gray
method

fine filamentous appendages

Compositon: protein: flagellin originate in cell membrane

3. Bacterial spore

a. Bacillus subtilis stained with grams stain

MICRO1 LAB 5
 bacterial
spore

Arrangement: bacilli in chains

highly refractile oval or spherical bodies w/in the vegetative cell

contains large amount of calcium dipicolinate which is believed associated with

the resistance of spores on heat

Present genus bacillus and genus clostridium

Bacillus subtilis representative organism: gram +

MICRO1 LAB 6
 demonstrate both central and subterminal spore

b. Clostridium tetani stained with Gram's stain

unaerobic bacteria = does not survive the presence of oxygen

terminal spore

draw 2-3 of these and label terminal spore

4. Slime layer and Capsule

Surface adherence strucutre

found just outside the cell wall surrounding the cell

difficult to visualize

MICRO1 LAB 7
 made of mucilaginous polysaccharide

surface cell adhesions

small amount = slime layer

definite layer = capsule

a. Streptococcus pneumoniae

stained with Anthony stain

don't use gram stain that is why special stain is used

↳ organism
to
clearly emphasize the capsule OF an

MICRO1 LAB 8
 diplococci = lanceolate doplococci

capsule is around the bacteria

clear halo of light surrounding the bacterial cell

mucilaginous substances which are polysaccharide in nature

special structure of s/ pneumoniae = capsule

don't forgot to label = capusle

b. Sarcina lutea

stained with dilute carbol fuchsin

MICRO1 LAB 9
 stained with India ink

slimy layer

whhat is the purpose of these special structures?

1.) contributes to the virulence of an
organism
2.) bacteria from recognition &
protects the

phagocytited immune
being
cells
from by

MICRO1 LAB 10
EX 3: HANGING DROP METHOD
↳ so we could visualize the organism

MATERIALS
1. Cultures of Gaffkya tetragena and Proteus Vulgaris
2. Concave Slides
3. Coverslips
4. Vaseline
PROCEDURE
Apply Vaseline or petroleum jelly on the edges of concavity of the slide to prevent
1 evaporation of the bacterial drop,

Take a loopful of the bacteria suspension and place it on the center of a clean
coverslip. Observe proper aseptic technique. (is done to prevent contamination
from the environment)
2
Proper aseptic technique is done to prevent contamination from the environment

ASEPTIC TECHNIQUE PROCEDURE

1 Hold the tube in one hand and the loop on the other.
2 Flame loop until red hot and remove the cotton plug.
Flame the mouth of the tube and insert the loop.
o Before you insert the wire loop into the tube, wait for 10-15 seconds after
3 heating because the heat will kill the organism

4 After taking a loopful of the bacterial suspension from the tube. You flame the mouth
of the tube again then you cover the tube with a cotton.
5 Taking your loopful of bacterial suspension, You place it on the center of your
coverslip.
6 And then you flame your loop until it’s red hot and place on the rack to cool.

back to the procedure

Carefully invert the prepared coverslip and apply it over the concavity making sure
that the drop of bacterial culture is in the center, and the drop is hanging inro the
concavity without touching the slide. Hence, “hanging drop”.
The hanging drop method is done so we can see the motility of the organism.

3 This is also why we use a concave slide so that there will be a space between the
coverslip and the slide and the drop is allowed to hang.
EX 3: HANGING DROP METHOD

This is also why we use a concave slide so that there will be a space between the
coverslip and the slide and the drop is allowed to hang.
But in the case that a concave slide is not available, an ordinary slide may be used
but ringed with Vaseline or pet jelly so that it forms a well which can elevate the
cover slip and give some space for the drop to hang.

The preparation is the studied under the Low Power Objective or LPO with the
diaphragm partly open. The best guide in locating the preparation is the edge of the
drop.
Why the edge of the drop is focused:
i. Better contrast is obtained due to the difference in refractive
4 index of the drop and the cover slip.
ii. Also, as the drop hangs, it thins toward the edge. This means
the edge would contain a lesser number of bacteria thus give
us a chance to see the motility clearer.
iii. Aerobic bacteria usually vomes toward the edge to gert more
oxygen for respiration.
After studying the preparation under the LPO, the LPO is raised very slightly before
switching to the HPO. This is because we have to prevent breaking the cover slip
or damaging the objective when the switch high power is made. Usually, the raising
of LPO is only done when we are using the old microscopes where the objectives
are the ones being raised. (Usually done on old microscopes but in our lab we use
new microscopes wo raising the LPO slightly before changing to HPO is not
necessary).
5

We will then observe the bacteria and draw the bacteria while taking note of its
6 form, arrangement, and movement. The direction of the movement of the bacteria
should be indicated using arrows.
EX 3: HANGING DROP METHOD

Now, all hanging drop preparations must be immersed in disinfectant or Sodium

7 Hypochlorite or bleach for 10-20 mins to kill the organism before cleaning.
TYPES OF MOVEMENTS EXHIBITED IN THIS ACTIVITY

Gaffyka tetragena Exhibits Brownian motion

Proteus vulgaris Exhibits True Motility

Is a to and fro motion of particles suspended in a liquid.

Is actually not true motility. It is a false motion. It only looks like it’s
moving due to the physical forces like the water molecules.
Representative organism: Gaffkya tetragena.
BROWNIAN
MOTION Discovered by British scientist Robert Brown in 1827. Using
microscope, he noticed pollen grains suspended in water jiggled.
Later determined that the motion was due to the action of collision to
the surrounding molecules
Later determined that the motion was due to the action of collision to
the surrounding molecules
Individual bacteria move across the slide with varying degree of
TRUE MOTILITY rapidity.
Representative organism: Proteus vulgaris
- Proteus vulgaris is peritrichous meaning the flagella is all over
the entire body or is uniformly distributed.
In this slide we can see side-by-side how a motile and non-
motile organism look when they move.

The motile organism would go on any direction while the

non-motile organism would look like they are just jiggling
in place or having a side-to-side motion.

cr: shanyu_twt
EX 4 : DIFFERENTIAL STAINS – GRAM STAIN

Making a bacterial smear from liquid media

4. After allowing
Objectives: When left standing for too the loop to cool for
long, organisms inside the at least 5 seconds,
- To familiarize the students with the culture tube tend to settle remove a loopful of
technique of Gram’s staining at the bottom. the organism.
- To familiarize the students with the Shaking the tube allows an
appearance of various bacteria when even distribution of the Purpose is also to avoid
stained using this procedure organisms in a liquid media contamination (from the air
- To familiarize the student with the procedure 1. Shake the culture Do not moisten cap on i.!! that may go into the tube)
of making bacterial smears from solid and tube from side to
liquid media side to resuspend
organisms. 5. Flame the mouth
GRAM STAIN of the culture tube
Principal stain used for again.
microscopic examination of
bacteria
Places bacteria into one of Last part of the Aseptic
Heating in this manner kills technique
two main groups:
the organisms that may be
1. gram-positive
on the wire loop. 6. Return the cap
Hucker’s Method (purple)
We can prevent and close it. Place
modified method 2. gram negative
-

contamination of the the tube on the test

(pink)
sa
gram stain culture tube. tube rack.
Provides a mechanism of
2. Heat the loop and First part of the aseptic
rapid presumptive wire until red hot. technique !!
identification of pathogens Flame handle
MATERIALS 7. Place the loopful
slightly also. of organisms in the
1. Cultures of Staphylococcus aureus, Branhamella
catarrhalis, Bacillus subtilis and Escherichia coli center of the target
2. Staining Reagents for Gram staining: circle on the slide.
Crystal violet
*size of smear =1 ½
Gram’s Iodine
cm
Acetone Alcohol
To make sure that the loop
Safranin This is to prevent 8. Flame loop again is sterile so that it is ready
3.Clean glass slides 3. Remove the cap contamination. before removing to be used for another
5. Alcohol lamps and flame the another loopful procedure.
Do not let the loop touch
mouth of the tube. from the culture or
4. Wire needles and Wire Loops the sides of the tube
Do not place the setting the
6. Tap water cap down the table. inoculating loop
aside.

PROCEDURE
EX 4 : DIFFERENTIAL STAINS – GRAM STAIN

Airdrying preserves the 5. Return the cap 4. Place slide on a Crystal violet serves as
morphology of the cells and close it. Place staining rack and primary stain
Smears must be dried the tube on the test flood the smear imparts color to all cells
9. The smear is before they are heat-fixed tube rack. with Crystal violet basic purple dye
then allowed to air solution for 1 stains all the cells purple
dry minute
5. Wash with tap Slides should be washed
water gently to prevent smears
from being washed out.
6. Two loopfuls or a Gram’s Iodine serves as
Making a bacterial smear from solid media drop of NSS are mordant.
First part of the Aseptic placed in the center Mordant is a
Technique. of the “target 6. Cover the smear
substance that
1. Heat the wire circle” with Gram’s Iodine
combines with a
needle until red for 1 minute and Mordant dye to fix it to a
hot. Flame handle then wash with tap -
"

dye material.
slightly also. water.
fixative
"
Enhances the
7. Place the needle
containing the Crystal violet
organism into the
target circle and 7. Tilt the slide and Wash the slide with tap
resuspend the add acetone water after 1 minute.
2. Remove the cap alcohol to
organism and allow Be sure to wash off the
and flame the decolorize the
them to mix into the slide properly as any
mouth of the tube. smear. Until no
NSS. remaining acetone alcohol
Do not place the more violet color may over decolorize the
8. Flame the needle
cap down the table. flows out the slide.
again before smear.
setting it aside. 8. Counterstain Basic Red
Avoid touching the sides of
with Safranin for 30 dye
3. After allowing the tube. seconds and wash Safranin Counterstain
the needle to cool
9. Allow the smear with tap water. for this
for at least 5
to air-dry before it procedure.
seconds, Remove a
is heat-fixed. 9. Blot dry the smears and examine them under
colony of the
the Oil Immersion Objective
organism.
Results:
Last part of the Aseptic GRAM STAINING PROCEDURE
technique (HUCKER’S METHOD)
4. Flame the mouth 1. Heat the slide Removes any grease
of the culture tube present . Pass slide 3-5
again. *Label the slide times over the flame.
before heating
2. Make thin smear allow to air dry
3. Heat-fix the kills the organisms and
smear binds them into the slide
EX 4 : DIFFERENTIAL STAINS – GRAM STAIN

Upon the addition of Crystal violet, all the cells stain

purple.
With the addition of Gram’s Iodine, the Crystal
Violet Iodine Complex is formed.
When decolorizing with acetone alcohol, the Gram Escherichia coli
(+) organisms retain their color. This is due to the bacilli
gram cocco
-

thick peptidoglycan wall being dehydrated by the

alcohol thereby trapping the CVI Complex.
On the other hand, the Gram (-) organisms lose
their purple color.
The Gram (-) organisms, instead of having a thick
peptidoglycan layer, have more
lipopolysaccharides on their cell wall which are
dissolved under the addition of acetone alcohol.
Thus, becoming colorless. Branhamella catarhallis
The addition of Safranin, which is the counter stain, ACID-FAST STAIN
gram Learning Objectives:
-

will stain the Gram (-) organisms pink or red.

1. To explain the principle and mechanism of Acid-
fast staining
2. To differentiate acid-fast from non-acid fast
bacteria
3. To provide an example of a clinical application of
Bacillus subtilis acid-fast stain

gram
t
strep
to bacilli
other commonly used stain
for light microscopic
examination of bacteria.
ACID FAST STAIN takes full advantage of the
Expected Results: waxy content of the cell
walls to maximize
Gram (+) organisms will appear dark purple detection.
Gram (-) organisms will appear pink to red specifically designed for a
Staphylococcus
subset of bacteria whose
epidermidis ALL COCCI ARE GRAM (+). except: Neisseria, principle cell walls contain long chain
t
staphylococcus
Moraxella catarrhalis fatty acids (mycolic acids)
gram
ALL BACILLI ARE GRAM (-)… except: render the cells resistant to
Mycobacteria, Nocardia, Corynebacteria, decolorization even with
Bacillus Mycolic acids acid alcohol decolorizers
*Draw the bacteria under Oil Immersion Objective
and Give the Staining Reaction and Morphology
stain poorly as gram (+)
Acid-fast bacteria bacilli
EX 4 : DIFFERENTIAL STAINS – GRAM STAIN

is the most commonly PROCEDURE minute. Then wash with water and allow to air dry.
encountered acid-fast 1. Before smears are stained, make sure to fix the DO NOT BLOT DRY.
bacteria smears. Methylene blue Counterstain used for this
Specifically, Heat fixing Common method procedure
Mycobacteria Mycobacterium If the lab has an incinerator, After this procedure, the acid-fast bacilli remain red
tuberculosis. you can perform heat fixing while the non-acid-fast bacilli and other cells take
as shown in the photo up the counterstain.
(the etiologic agent of 5. When smears are dry, examine microscopically.
tuberculosis.) and.
Bacteria lacking cell walls Scan slides at LPO
Can also be done by
fortified with mycolic acids Screen them at HPO
passing the flame under the
cannot resist decolorization Confirm all suspicious organismsat OIO
slide
Non-acid-fast with acid alcohol From a sputum sample with a 3+ grading of acid
2. Flood smear with carbolfuschin and heat to
trait of most other clinically fast bacilli
almost boiling by performing the procedure on
relevant bacteria
electrically heated platform or by passing the flame
PROCEDURE
of a Bunsen burner or Alcohol lamp underneath the
Classic acid-fast staining
slides on a metal rack. The stains on the slides
method should steam. Allow slided to sit for 5 minutes after
Ziehl-Neelsen Hot method heating and do not allow them to dry out. Wash the
Requires heat to allow slides with distilled water. After, drain off excess
primary stain to penetrate liquid.
the wax-containing cell wall Primary stain of acid fast
Carbolfuschin stain

Because their tap water is

contaminated with It is easy to locate the bacilli at this rate. But
Mycobacterium gordonae imagine if there’s only one or two bacilli present
Use of distilled Tap water may contain and you’re reading is the only basis for the patient
water acid-fast bacilli but here in to be diagnosed of laboratory-confirmed
the Ph our tap water are Pulmonary tuberculosis.
processed so we can use From a perichardial fluid
Before smears are stained, all cells in the slide are tap water. sample
colorless. 3. For decolorization, flood slides with acid alcohol.
After addition of primary stain, both acid fast and Allow reagent to sit for one minute. Check to see if *Snapping and slipping
non-acid fast bacteria take up the stain and stain no more red-color runs off the surface when the diplobacilli
red. slide is tipped. Add more decolorizer if you Poorly collected sputum
Just like Gram staining, the decolorizer sets them continue to see a red dye. Wash thoroughly with
sample with many
apart water and remove the excess.
epithelial cells and few
The acid-fast bacteria is resistant to decolorization Contains 3% Hcl in 95% pus cells
Primary stain from non-acid fast bacteria washes Acid alcohol ethanol
off.
When the counter stain is added, all non-acid-fast 4. Flood slides with methylene blue and allow the
bacteria as well as other cells take up the stain. stain to remain on the surface of the slide for 1
The acid-fast bacteria remain red.
EX 4 : DIFFERENTIAL STAINS – GRAM STAIN

Center is filled with non-

acid-fast bacilli

PROCEDURE -

heat is not required

stain
Cold Method for
Kinyoun because primary
Does not require the contains high concentration
use of heat of phenol
Primary stain contains
Kinyoun higher concentration
of phenol (why heat is
not required) for
intracellular
penetration.

cr: shanyu_twt
EX 5 : METHODS OF OBTAINING PURE CULTURE

STREAK PLATE METHOD

MATERIALS
1. Cotton swab
2. Nutrient agar
3. Sterile Normal Saline Solution
4. Wire Loop
PROCEDURE 16. From the same colony, obtain an inoculum,
1. Moisten a sterile cotton note: do not forget to inoculate another NA plate and do 4 quadrant
swab with Sterile NSS flame the mouth of streaking.
the NSS tube before 17. Incubate the plate for 37°C for 24 hours. 9. After 24 hours of incubation, look at the plate and
and after this note the size of the colonies in and on the medium.
procedure 18. After 24 hours, examine the plate
2. Take a swab sample note: in the video
they swiped the area
in concentric circles
3. Roll the cotton swab in a small area of the first
quadrant.
4. Heat the wire loop until red hot and let it cool.
5. Using a sterile wire loop, spread the inoculum by
streaking back and forth over the first quarter of the
agar surface
6. Heat wire loop again and let it cool
19. Do a Gram stain of the isolated colonies
7. Turn the plate 45° counterclockwise for the 2nd
quadrant POUR PLATE METHOD
8. Start streaking across a portion of the 1st quadrant MATERIALS
2-3 times 1. Pre-diluted suspension
9. Continue streaking the 2nd quadrant but do not 2. Sterile Petri Dish
allow the succeeding strokes to touch the 1st 3. Sterile Pipette
quadrant. 4. Test tube with melted Nutrient Agar
10. Heat wire loop again and let it cool. PROCEDURE
11. Repeat the procedure for the 3rd and 4th note: do not forget to
quadrants, with heating and cooling the loop in 1. Using a sterile pipette, flame the mouth of
between. remove 1 ml of the the tube containing
12. On the 4th quadrant, do not allow the final strokes suspension. the suspension
to touch any streaked area. before and after
13. Incubate the plate for 37°C for 24 hours. inserting the pipette.
14. After 24 hours of incubation, examine the plate 2. Discharge the suspension note: flame the lower
in a petri dish part of the pipette
quickly before putting
it away
3. Get the melted tube of agar and allow it to cool to
the point that it can be held comfortably in the hand.
4. Remove the cover and flame the mouth of the tube.
5. Lift the lid and pour the agar into the petri dish.
6. Gently swirl the petri dish on the table top ti evenly
distribute the agar.
7. Allow the agar to harden.
8. Incubate the plate upside down at 37°C for 24
hours.
15. Do a Gram stain for the predominant isolated
colony.
EX 5 : METHODS OF OBTAINING PURE CULTURE

10. Using a Quebec colony counter, count the

colonies on the plate.
 EX6 - EFFECTS OF PHYSICAL AGENTS ON MICROORGANISMS

Organisms Used: Bacillus subtilis, Escherichia coli

Materials: Filter paper impregnated w/ the organisms, Nutrient Broths, Forceps, alcohol lamp

Organisms Control Boiling 62oC Autoclave Hot Air

Bacillus subtilis Turbid Turbid Turbid Clear Clear
Escherichia coli Turbid Clear Clear Clear Clear

Organisms Medium of Choice Classification Medium

Bacillus subtilis Nutrient Broth Nutritive/ Supportive/ Non-Selective
Escherichia coli Nutrient Broth Nutritive Media / Supportive/ Non-Selective

RESULTS & INTERPRETATION:

REMEMBER: if observation is TURBID, bacteria is NOT KILLED.

if observation is CLEAR, bacteria is KILLED.

Example:
Reading: Turbid
Interpretation: Organism is NOT KILLED

Reading: Clear
Interpretation: Organism is KILLED
 EX7 – ACTION OF DISINFECTANTS ON MICROORGANISMS

Organisms used: Escherichia coli

Materials: culture of Escherichia coli, 5 lactose broths (per disinfectant) in Durham’s fermentation tubes, timer, disinfectants (5% phenol, betadine, merthiolate, 5% lysol, 1:1000 mercuric chloride,
95% isopropyl alcohol, 3% hydrogen peroxide, 1% sodium hypochlorite, 70% ethyl alcohol, 1:1000 zephiran chloride), wire loop, sterile pipette, bulb aspirator, alcohol lamp

5% PHENOL BETADINE MERTHIOLATE 5% LYSOL 1:1000 MERCURIC CHLORIDE

MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION:
Damage the cell membrane Protein Oxidation Protein Coagulation Damage the cell membrane Enzyme Inactivation
95% ISOPROPYL ALCOHOL 3% HYDROGEN PEROXIDE 1% SODIUM HYPOCHLORITE 70% ETHYL ALCOHOL 1:1000 ZEPHIRAN CHLORIDE

MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION: MECHANISM OF ACTION:
Protein Coagulation Protein Oxidation Protein Oxidation Protein Coagulation Damage the cell membrane

DAMAGE THE CELL MEMBRANE PROTEIN OXIDATION PROTEIN COAGULATION ENZYME INACTIVATION
5% Phenol Betadine Merthiolate
5% Lysol 3% Hydrogen Peroxide 95% Isopropyl Alcohol Mercuric Chloride (1:1000)
Zephiran Chloride (1:1000) 1% Sodium Hypochlorite 70% Ethyl Alcohol

RESULTS & INTERPRETATION:

REMEMBER: if observation is NC or no change, Gram Staining is ND or not done.

Example:
Reading: No change
Interpretation: E.coli (name of the organism) is killed at 5 minutes (indicate the least time it is killed)

DISINFECTANT CONTROL 5’ 10’ 15’ 30’

5% Phenol AG NC NC NC NC
Betadine AG NC NC NC NC
Merthiolate AG NC NC NC NC
5% Lysol AG NC NC NC NC
Mercuric Chloride (1:1000) AG NC NC NC NC
95% Isopropyl Alcohol AG NC NC NC NC
3% Hydrogen Peroxide AG NC NC NC NC
1% Sodium Hypochlorite AG NC NC NC NC
70% Ethyl Alcohol AG NC NC NC NC
Zephiran Chloride (1:1000) AG NC NC NC NC

EX8 - ANTIMICROBIAL SUSCEPTIBILITY TESTING: DIFFUSION TEST PROCEDURE:

Organisms used:
Enterobacter cloacae Escherichia coli Staphylococcus aureus Shigella dysenteriae
Klebsiella pneumoniae Pseudomonas aeruginosa Proteus vulgaris Salmonella typhi
Salmonella enteritidis Salmonella Paratyphi A

Materials:
• STANDARD PREP: Stock culture of the 10 bacteria inoculated on TSA Slant, 0.5 McFarland Standard (aka Barium Sulfate Comparison Standard), Black background, NSS, Inoculating
loop/wire needle, Alcohol lamp, Screw capped tubes
• INOCULATION: Standardized bacterial suspension, Test tube rack, alcohol lamp, MHIA Plate Mueller-Hinton Agar, sterile cotton swab, infectious waste container
• APPLICATION OF ANTIBIOTIC DISC: Forceps, Alcohol lamp, 95% absolute alcohol, Antibiotic discs
• FOR READING: Ruler or caliper (measure diameter in mm)
•
ANTIBIOTIC DISC POTENCY/CONCENTRATION ABBREVIATION MECHANISM OF ACTION
Chloramphenicol 30 ug C30 Inhibition of protein synthesis

Penicillin G 10 ug P10 Inhibition of bacterial cell wall synthesis

Trimethoprim Sulfamethoxazole 25 ug (Trimethoprim-1.5 ug; SXT25 Target folic acid pathway
Sulfamethoxazole – 23.75 ug)
Gentamicin 10 ug CN10 Inhibition of protein synthesis
Ampicillin 10 ug AM10 Inhibition of bacterial cell wall synthesis

RESULTS
Antimicrobial Agent Disc Inhibition zone diameter to the nearest mm
Potency Resistant Intermediate Sensitive Your Interpretation
result
Ampicillin Enterobacteriaceae 6 mm Resistant
& enterococci 10 ug 11 or less 12-13 14 or more
Ampicillin Staphylococci 10 ug 20 or less 21-28 29 or more 6 mm Resistant
Ampicillin Haemophilus 10 ug 19 or less 20 or more
Chloramphenicol 30 ug 12 or less 13-17 18 or more 17 mm Intermediate
Gentamicin 10 ug 12 or less 13-14 15 or more 11 mm Resistant
Penicillin G 10 ug 20 or less 21-28 29 or more
Staphylococci
Penicillin G 10 ug 11 or less 12-21 22 or more 6 mm Resistant
Other Organism
Trimenthoprimsulfamethoxazole 1.25 + 10 or less 11-15 16 or more 6 mm Resistant
(SXT) 23.75 ug
= 25 ug
 Kani kay di na guro ni mugawas pero mata mataha lang nya basin diay kay part raba nis exercise

Organism Agar Temp Atmosphere Time of incubation

Neisseria gonorrhoeae GC agar plus cysteine-free 35oC Room air 20-24 hr
supplements
Streptococcus pneumoniae Mueller-Hinton Agar (MHA) 35oC 5-7% CO2 20-24 hr

Haemophilus influenzae Haemophilus test medium 35oC 5-7% CO2 16-18 hr

Neisseria meningitidis Mueller-Hinton Agar 35oC 5-7% CO2 20-24 hr

(MHA) plus 5% sheep blood
 EX9 – Staphylococcus
Organisms used: Staphylococcus aureus, Staphylococcus Epidermidis
Materials: Stock cultures, BA, TSA, Mannitol Agar, Plasma (medium for Coagulase test), NSS, Hydrogen peroxide, Inoculating wire loop and wire needle, Alcohol lamp

Staphylococcus aureus Staphylococcus epidermidis

GROWTH ON BLOOD AGAR

HEMOLYSIS ON BLOOD AGAR

GROWTH ON TSA
 GRAM STAIN

Interpretation Results

Slide Coagulase Test

Positive presence of white precipitate or
agglutination within 10-15 sec
Negative Smooth and milky homogenous
mixture

Tube Coagulase Test

Positive Complete clotting observed
Weakly positive A small amount of clot/
coagulum
Negative No clot/coagulum formation
 Mannitol Fermentation Test
Positive A (Acid)
Negative No change

Catalase Test
Positive Immediate appearance of gas bubble
Negative No gas bubble formation
 MICRO LAB EXERCISE NO. 10 & 11 - ALPHA AND BETA-HEMOLYTIC STREPTOCOCCI

MATERIALS

Specimen: Throat swab specimen

Incubation settings: 37 degrees Celsius in candle jar; Examine after 24 hours; 5-10% CO2

STREAKING – PRIMARY PLATE TO ½ BA: multiple interrupted streaking without heating in between

SENSITIVITY TESTING: Vertical line across ½ of BA plate, streak closely across the vertical line (pls specify the term)

F5 | !
 RESULTS/OBSERVATIONS/INTERPRETATION

ALPHA HEMOLYTIC STREPTOCOCCI BETA HEMOLYTIC STREPTOCOCCI

Colonial Morphology: small, gray, round, transparent, Colonial Morphology: pinpoint, transparent to translucent,
glistening, mucoid, entire edges convex, circular, entire, matte, shiny or glossy
Hemolytic Property: Alpha hemolysis (partial hemolysis, green) Hemolytic Property: Beta- Hemolysis (complete hemolysis) - the
Remember: PAG city area appears lightened (yellow) and transparent
Partial-Alpha-Green Remember: CeBu Yarn?
Complete-Beta-Yellow

Colonial Morphology
and Hemolytic Property

Streptococcus pneumoniae streptococcus pyogenes

Staining Reaction: Gram (+) Staining Reaction: Gram (+)

Morphology: Diplococci (lanceolate) Morphology: Streptococci

Gram Staining

Reading: positive (+)

Staining Method: Anthony Staining Method
Specimen: Culture from skimmed milk
Principle:
Capsule Staining
- As a capsule staining procedure, the primary stain is
Crystal Violet and will be taken up by all cells.
- A 20% Copper sulfate solution will serve as the
decolorizing agent and counterstain

F5 | !
 Reading: ACID
Interpretation: Inulin fermenter

SIDE NOTE: If no change before and after inoculation (remains

orange)
Reading: Negative
Interpretation: Inulin is not fermented
PRINCIPLE:
- Differentiates S. pneumoniae from other Streptococci
- S. pneumoniae hydrolyzes inulin
- Orange --> yellow due to acid formation
Inulin Fermentation - Produces acid without gas

Reading: Clear
Interpretation: Bile soluble
Bile Solubility Test
SIDE NOTE: If still turbid after addition of reagent
Reading: Turbid
Interpretation: Bile insoluble
F5 | !
 PRINCIPLE:
- Distinguishes S. pneumoniae from other alpha-
hemolytic Streptococci
- 10% Sodium deoxycholate lyses S. pneumoniae while
other Streptococci doesn’t lyse
- Bile soluble --> presence of enzyme autolytic amidase
(cleaves the bond between alanine and muramic acid
in the peptidoglycan)

Reading: 15 mm zone of inhibition

Interpretation: susceptible to optochin
PRINCIPLE:
- Zones ≥ 14 mm surrounding a 6 mm diameter disc
- Zones ≥ 16 mm surrounding a 10 mm diameter disc are
presumptive of S. pneumoniae
Remember OVeRPaS
Optochin: Viridans – Resistant
Pneumoniae - Sensitive

Optochin Sensitivity Test

F5 | !
 Reading: 20 mm zone of inhibition
Interpretation: susceptible to Bacitracin
PRINCIPLE:
- Sensitive to Group A Streptococci
- Any zone of growth inhibition is indicative of Bacitracin
susceptibility
Remember BRaS
Bacitracin: Resistant – Agalactiae
Sensitive – Streptococcus pyogenes

Bacitracin Susceptibility
Test

F5 | !
F5 | !
 Antimicrobial Agent Disc Inhibition zone diameter to the nearest mm
Potency Resistant Intermediate Sensitive Your Interpretation Slide Coagulase
result Principle
Ampicillin Enterobacteriaceae • Bound coagulate is bound to the bacterial cell wall and reacts directly with
& enterococci 10 ug 11 or less 12-13 14 or more fibrinogen.
Ampicillin Staphylococci 20 or less 21-28 29 or more 6mm R • This results in an alteration of fibrinogen so that it precipitates on the
Ampicillin Haemophilus 19 or less 20 or more
staphylococcal cell causing the cells to clump when a bacterial suspension
Chloramphenicol 30 ug 12 or less 13-17 18 or more 23mm S
is mixed with plasma.
Gentamicin 10 ug 12 or less 13-14 15 or more 12mm R
Penicillin G 10 ug 20 or less 21-28 29 or more 6mm R
• This does not require coagulase-reacting factor.
Staphylococci
Notes
Penicillin G 11 or less 12-21 22 or more ▪ Clumping factor directly converts fibrinogen to fibrin causing agglutination
Other Organism ▪ Heavy suspension of organism is made on glass slide and mixed with drop
Trimenthoprimsulfamethoxazole 1.25 or 10 or less 11-15 16 or more 20mm S of plasma
(SXT) 23.75 ug ▪ Agglutination indicates a positive test:
▪ Indicates Staphylococcus aureus
Antibiotic Ampicillin Chloramphenicol Trimethoprim Gentamicin Penicillin
▪ Some species of coagulase negative staphylococcus can be positive
AM10 C30 Sulfamethoxazole CN10 P10 ▪ Negative results should be confirmed with tube coagulase test.
SXT Observations
GRP ORGANISM Mm Int. Mm Int. Mm Int. Mm Int. Mm Int. 1. Coagulase positive: Macroscopic clumping in 10 seconds or less in
1 E.cloacae 6mm R 10mm R 19mm S 15mm S 6mm R coagulated plasma drop and no clumping in saline or water drop.
2 E.coli 6mm R 28mm S 26mm S 16mm S 6mm R 2. Coagulase-negative: No clumping in either drop
3 K.pneumoniae 6mm R 31mm S 6mm R 16mm S 6mm R Interpretations
4 P.aeruginosa 26mm S 17mm I 6mm R 11mm R 6mm R • Slide coagulase test is the main method used to identify S. aureus in
5 P.vulgaris 6mm R 22mm S 18mm S 12mm R 6mm R clinical laboratories but it has some limitations.
6 S.aureus 6mm R 23mm S 26mm S 12mm R 6mm R 1. About 15% of ordinary strains of S. aureus and many more of MRSA
7 S.dysenteriae 18mm S 32mm S 26mm S 12mm R 8mm R give negative reactions.
8 S.enteritidis 21mm S 26mm S 21mm S 17mm S 22mm S 2. Few species of coagulase-negative staphylococci give positive reactions.
Procedure
1. Divide the glass slide into C control and T test
2. Place a small drop of NSS on both sides.
3. Make a heavy suspension with the isolated colonies on BA.
Materials 4. Add loopful of plasma to T and observe clumping within 10 seconds.
1. Staphylococcus aureus and staphylococcus epidermidis. 5. Test for Cell bound coagulase.
2. Blood agar plates, trypticase soy agar, mannitol agar. Tube Coagulase
3. Plasma (medium for coagulase test) Principle
4. H2O2; Sterile NSS • Isolates not producing clumping factor should be test for the ability to
Preparation produce extracellular coagulase (free coagulase) causing clotting of plasma.
1. Make a smear and do a gram stain of the organisms.
• Involves the activation of plasma coagulase-reacting factor (CRP) which is
2. BA plate = 4 quadrant streaking
a modified or derived thrombin molecule to form a coagulase-CRP
3. TSA Slant = simple streaking,
complex.
4. Incubation – 37C for 24 hours
• This complex in turn reacts with fibrinogen to produce the fibrin clot.
• Colonial characteristics on BA
Procedure
• Hemolytic property on BA 1. Small test tube with 0.5ml fresh human plasma.
• Pigment Production on TSA Slant 2. Inoculate the plasma with a loopful of bacteria from BA.
3. Incubate at 37C and observe clotting at intervals of 30 minutes for 4 hours.
 4. Test for free coagulase
Observation
• Read as positive any degree of clot formation. Often the plasma is
converted into a stiff gel that remains in place when the tube is tilted or
inverted, but sometimes clots are seen floating in the fluid.
1. Coagulase Positive: Clot of any size eg. Staphylococcus aureus
2. Coagulase Negative: No clot (plasma remains wholly liquid or shows
only a flocculent or ropy precipitate). eg. Staphylococcus epidermidis
Procedure
1. Add 1mL of H2O2 into TSA slant
2. Observe for immediate appearance of gas bubbles.
Results
• The positive test is demonstrated by the immediate appearance of bubbles.
• The appearance of one or two bubbles represents a weak reaction.
• A negative test is represented by no bubbles or a few bubbles after 20 s.

Quality Control: With each batch of the tests include tubes with known coagulase-
positive and coagulase-negative cultures as controls, and a tub of unseeded diluted
plasma to confirm that it does not clot spontaneously.

Mannitol Fermentation
Principle
• Mannitol salt agar contains beef extract and proteose peptone, which makes
it very nutritious as they provide essential growth factors and trace nutrients Materials
such as nitrogen, vitamins, minerals and amino acids essential for growth. 1. Stock cultures of Streptococcus pneumoniae and Streptococcus viridans.
2. Blood agar plates
• The medium contains 7.5% concentration of sodium chloride which results
3. Inulin Agara
in the partial or complete inhibition of bacterial organisms other than
4. Skimmed Milk
staphylococci.
5. Sug Optochin disc (Ethylhydrocupreine HCL)
• Mannitol is the fermentable carbohydrate source, fermentation of which
6. 10% Solution of Sodium Deoxycholate
leads to acid production.
7. 1% Aqueous crystal violet
• Indicator – the pH indicator phenol red is red at neutral pH but turns yellow
8. 20% Copper Sulfate
at pH <6.8. It also changes to magenta or hot pink at pH >8.4. 9. Sterile Normal Saline Solution
Procedure
10. Inoculating wire loop and wire needle, alcohol lamp.
1. Get an inoculum from TSA using a wire needle and stab the agar until few
PROCEDURE
mm from the bottom
First Meeting
2. Incubate at 37C for 24 hours.
1. Make a smear from the stock culture of alpha-hemolytic Streptococcus
3. Observe color change (Acid – Pink, No Change – Red-Pink)
assigned to the group. Do gram stain.
Result
2. From the same source, inoculate a BA plate and a tube of skimmed milk.
• (+) – Yellow colored solution – ACID Production 3. Incubate both media at 37C inside a candle jar.
• (-) – Red Colored Solution – NO CHANGE 4. After 24 hours incubation, examine and describe the colonies growing on
Catalase Production BA.
Principle • Take note of their colonial characteristics and hemolytic property.
• Bacteria capable of synthesizing the enzyme catalase hydrolyze hydrogen Second Meeting (Capsule Staining – Anthony Method)
peroxide into water and gaseous oxygen which results in the liberation of 1. Make a thin even smear of a culture in skimmed milk spreading with a glass
gas bubbles. slide.
• Metabolic activity of aerobic and facultative anerobic microorganisms 2. Air dry, do not fix with heat.
produce toxic by products like hydrogen peroxide and superoxide radical. 3. Stain with 1% aqueous crystal violet for 2 minutes.
• These products are toxic to the organisms and might even result in cell lysis 4. Wash with a solution of 20% copper sulfate.
if not broken down. In the case of pathogenic organisms different 5. Air dry in a vertical position and examine under oil immersion objective.
mechanisms are found that break down these products to non-toxic • The capsule is unstained against a purple background
substances. • The cells are deeply stained.
• The production of catalase protects the organisms against the lethal effect With the colonies growing on BA, perform the following:
of hydrogen peroxide accumulated at the end of the aerobic metabolism. Make a smear and do gram stain
Capsule Staining – Anthony Method
Principle
• Positive staining method used in capsule staining.
• The smear or sample is treated with a hypertonic solution (copper sulfate)
for the purpose of obtaining an ionic difference that results in the diffusion
of a stain towards the outer surface of the cell.
• Copper sulfate is used as a decolorizing agent.
• Copper sulfate washes the purple primary stain out of the capsular material
without removing the stain bound to the cell wall. At the same time. The
decolorized capsule absorbs the copper sulfate, and the capsule will now
appear blue in contrast to the deep purple color of the cell since the capsule
is non-ionic unlike the bacterial cell. Therefore, washing out the primary
stain out of the capsule but not from the cell wall.
Bile solubility Test
Principle
• The bile solubility test is used to identify and distinguish S.pneumoniae
from alpha-hemolytic Streptococcus spp.
• The test may be performed using a cell suspension on a slide or in a tube or
by adding the reagent directly to the colony.
• Principle of the test is the lysis of pneuomococcal cells when sodium
deoxycholate (bile salts) is applied to the colony. Under specific conditions
of time and temperature but other streptococci do not lyse.
• The pneumococcus has an intracellular autolytic enzyme, an amidase,
which causes the organisms to undergo rapid autolysis when cultivated on
an artificial medium.
• The bile salts alter the surface tension of the medium and cause cell
membrane rearrangement.
Procedure
1. To a tube containing 2mL of NSS, add colonies until you have a turbid
suspension.
2. Divide the suspension into 2 test tubes.
3. Mark one tube as CONTROL
4. To the other test tube, add a few drops of 10% solution of sodium
deoxycholate.
5. Observe for clearing or lysis which occurs in 5-10 minutes. Compare the
turbidity with that of the control tube.
Results
• (+) – Clear Solution
• (-) – Turbid Solution
Limitations
1. Bile salts will not induce clearing of a killed culture or one that is too acid.
Therefore saline suspensions of young cultures are used.
2. The test should not be performed on old cultures, as the active enzyme may
be lost.
3. Normal autolysis of S. pneumoniae may be inhibited by a high
concentration of bile salts being used. Evaporation may cause the reagent to
become more concentrated, therefore affecting the test.
4. When performing the bile solubility tube test using saline or unbuffered
broth, it is essential to adjust the pH to neutral before adding the reagent in
order to avoid false negative reactions.
5. When testing using the plate method, care must be taken not to dislodge the
colony being tested, therefore leading to false positive results.
Inulin Fermentation
1. Using a sterile inoculating wire needle, inoculate an inulin agar by stabbing
the medium until a few mm from the bottom.
2. Incubate at 37C and read the result in 24-28 hours. Observe the color
change I in the medium.
Optochin sensitivity test
Priciple
• Optochin (ethylhydrocupreine hydrochloride) is a chemical and is
completely soluble in water.
• Optochin is an antibiotic that interferes with the ATPase and production of
adenosine triphosphate (ATP) in microorganisms.
• Optochin is used in the presumptive identification of alpha-
hemolytic Streptococcus pneumoniae.
• The chemical tests the fragility of the bacterial cell membrane and causes S.
pneumoniae to lyse due to changes in surface tension. S. pneumoniae is a
gram-positive lanceolate diplococcus, but can also occur as single cocci or
in short chains of cocci. S. pneumoniae is a fastidious bacterium, growing
best at 35-37°C with ~5% CO2.
• Differentiating pneumococci from viridans streptococci is difficult as young
pneumococcal colonies appear raised, similar to viridans streptococci.
• Optochin sensitivity allows for the presumptive identification of alpha-
hemolytic streptococci as S. pneumoniae, although some pneumococcal
strains are optochin-resistant.
• Other alpha-hemolytic streptococcal species are optochin-resistant. For the
optochin susceptibility test, the Optochin impregnated disk is placed on a
lawn of the organism on a sheep blood agar plate, allowing the antibiotic to
diffuse into the medium.
• The antibiotic inhibits the growth of a susceptible organism, creating a
clearing, or zone of inhibition, around the disk.
• A zone of 14mm or greater is considered susceptible and presumptive
identification for Streptococcus pneumoniae.
Procedure
1. Inoculate a BA plate heavily by streaking closely with 2 or 3 colonies as
inoculum.
2. Place an optochin disc at the center of the inoculated plate.
3. Incubate the plate aerobically at 37C.
4. Read the result 24 hours after incubation. Observe for zones of growth
inhibition surrounding the disc.
Result
• Optochin Susceptible
• Zones > 14mm surrounding a 6mm diameter disc
• Zones > 16mm surrounding a 10mm diameter disc
• considered positive and presumptive identification of
streptococcus pneumoniae.
• Optochin Resistant:
• No zone of inhibition around the disk.
• If the zone of inhibition is less than 14 mm, further testing (bile
solubility or serology) should be done for the identification of
other strains of Streptococcus pneumoniae.
Limitations
1. Streptococcus pneumoniae isolates should be incubated in a CO2enriched
environment, as some isolates will grow poorly or not at all.
2. Optochin susceptibility is a presumptive test only. It is recommended that
further biochemical tests be performed for complete identification.
3. Any zone of inhibition less than 14 mm is questionable for pneumococci;
the strain is identified as pneumococcus with confirmation by a positive
bile-solubility test or serology can be performed.
Materials
1. stock cultures of Group A beta-hemolytic streptococci and non-group A
beta-hemolytic streptococci.
2. Blood Agar Plate
3. 0.04U Bacitracin Disc
4. Forceps, inoculating the wire loop, alcohol lamp.
Procedure
1. Make a smear from the stock culture of beta-hemolytic streptococci
assigned to the group. Do Gram Stain
2. From the same source, inoculate a BA plate using a wire loop.
• do the first quadrant streaking as usual but make 2-3 stabbings at the end
of the last line.
• 2nd, 3rd and 4th quadrant without stabbing at the end of the last line.
• Incubate the plate at 37C and take not of the colonial and hemolytic
properties of the organism.
Bacitracin Test
Principle
• The growth of Group A beta-hemolytic Streptococci on blood agar is
inhibited by 0.04 units Bacitracin disc.
• Micrococci and streptococci are also inhibited by 0.04 units disc, while all
coagulase-negative staphylococci are resistant.
• Bacitracin susceptibility test discs are filter paper discs impregnated with
0.04 units of Bacitracin. The impregnated disks are then placed on the agar
where allows the antimicrobial to diffuse through the medium and inhibit
the growth of the organism.
• The test result is evaluated after incubation on the basis of the zone of
inhibition observed around the disks.
• If the growth of the organism is observed up to the edge of the disk, it is
deemed resistant, whereas the presence of a circular zone around the disk
represents inhibition and susceptibility.
• Bacitracin discs can save considerable time, labor, and materials if used as a
screening test before serological grouping.
• It has been observed that Group A Streptococci were more sensitive to
Bacitracin than beta-hemolytic strains of other groups.
• Hence Bacitracin susceptibility test via antimicrobial disks is suggested as a
rapid diagnostic agent for Group A Streptococci.
Procedure
1. From the same stock culture, obtain a heavy inoculum (3-4 colonies) and
inoculate ½ of another BA plate by streaking as follows:
• Make a vertical line across ½ of the BA plate. Streak closely across this
vertical line with the loop in a vertical position.
2. With the sterile forceps, place bacitracin disc on the center of the inoculated
area.
3. Incubate the BA plate aerobically at 37C
4. Read the results in 24 hours after incubation and examine growth inhibition
around the bacitracin disc.
• Any zone of growth inhibition is indicative of bacitracin susceptibility.
Results
Zone of Inhibition Media Result
6 mm or less Resistant
10 mm or more Susceptible
Blood Agar Tests are repeated as it
Between 6 mm and 10 mm indicates probable
susceptibility
Limitations
• Old blood agar plates that have dried should not be used for susceptibility
testing as the diffusion of antibiotics on such agar is reduced, which might
result in false-negative results.
• Different zone of inhibition sizes might be observed with different
concentrations of Bacitracin; thus, differential disks (0.04 U) should be used
instead of sensitivity disks (10 U).
• Light inoculum might result in a false zone of inhibition, and thus,
sufficient inoculum resulting in confluent growth should be used.
• For further confirmation, serological testing like latex agglutination tests
should be performed to obtain accurate confirmation.
• Staphylococci show no zone of inhibition around the bacitracin 0.04-U disk
on BAP. Zone of inhibition of sizes greater than7 mm but less than the 10-
mm breakpoint may be obtained for Micrococcus if incubation is not a full
24 h or MH agar is used.

Materials
1. Cultures of Neisseria meningitidis and Neisseria gonorrhoeae
2. Stock culture of Neisseria sicca and Branhamella catarrhalis
3. Chocolate agar
4. Nutrient Agar
5. Glucose, Maltose, sucrose and fructose agars.
6. Oxidase reagent (1% p-aminodimethylaniline oxalate)
7. Inoculating wire loop and wire needle, alcohol lamp.
Pathogenic Neisseria
Procedure
1. Study and observe the gross and microscopic appearance of N. meningitidis and
N.gonorrhoeae
2. Observe the fermentation reactions of N. meningitidis and N.gonorrhoeae in
glucose, maltose, sucrose and lactose agars
Non-Pathogenic Neisseria
Procedure Meeting 1
1. Make a smear and do gram stain.
2. Inoculate the CA and NA by 4-quadrant streaking.
3. Glucose, maltose, sucrose and lactose are inoculated by stabbing until a few mm
from the bottom.
4. Incubate all the inoculated media at 37C inside the candle jar except NA which is
incubated aerobically.
5. After 24-hour incubation, examine the plates and describe the colonies isolated.
6. Observe for any color change in the inoculated sugars.
Procedure Meeting 2
1. Make a smear of an isolated colony on NA plate and gram stain.
Oxidase Test
Principle
• Cytochrome containing organisms produce an intracellular oxidase enzyme. This
oxidase enzyme catalyzes the oxidation of cytochrome c.
• Organisms which contain cytochrome c as part of their respiratory chain are
oxidase-positive and turn the reagent blue/purple.
• Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the
reagent, leaving it colorless within the limits of the test, and are oxidase-negative.
• Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an
iron containing haemoprotein).
• Both of these catalyse the transport of electrons from donor compounds (NADH) to
electron acceptors (usually oxygen).
• The test reagent, N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride acts
as an artificial electron acceptor for the enzyme oxidase.
• The oxidised reagent forms the coloured compound indophenol blue.
• The cytochrome system is usually only present in aerobic organisms which are
capable of utilising oxygen as the final hydrogen receptor.
• The end product of this metabolism is either water or hydrogen peroxide (broken
down by catalase).
Procedure
1. Perform the test on the colonies growing on the NA plate by adding a few drops of
1% solution of p-aminodimethylaniline oxalate.
2. Observe the colonies turn pink, red, purple and black in 10-30 seconds.
Result Interpretation of Oxidase Test
• Positive Result
o Development of a deep purple-blue/blue colour indicates oxidase production
within 5-10 seconds.
• Negative Result
o No purple-blue colour/No colour change.
Limitations
1. The reagents used in the oxidase test have been shown to auto-oxidize, so it is very
important to use fresh reagents, no older than 1 week.
2. Both bacteria and yeast grown on media containing high concentrations of glucose
show inhibited oxidase activity, so it is recommended to test colonies grown on
media without excess sugar, such as nutrient agar. Tryptic soy agar is also an
excellent media.
3. Bacteria grown on media containing dyes may give aberrant results.
4. The test reagents will effectively kill the microorganisms, so sub-culturing should be
done prior to adding any reagent to an active culture.
5. The oxidase test can be used in the presumptive identification of Neisseria and in
the differentiation and identification of gram-negative bacilli. Oxidase-positive
organisms should be examined by gram stain to determine morphology and gram
reaction. Additional biochemical tests are recommended for complete identification.
6. Use of a nichrome or other iron containing loop may yield false-positive reactions.
Platinum loops are recommended.
7. Most Haemophilus are oxidase-positive. Less sensitive strips or reagents may yield
false-negative results.
8. Oxidase reactions of gram-negative bacilli should be determined on non-selective
and non-differential media to ensure valid results. Also, colonies taken from media
containing high levels of glucose may give false-negative reactions.
9. It is recommended to use colonies that are 18-24 hours old. Older colonies will
produce weaker reactions.
10. Any color changes appearing after 20 seconds should be disregarded.
Sugary Fermentation Tests
Principle
• Carbohydrate fermentation is the process by which the microorganism utilizes to
produce energy in the form of ATP, the ultimate energy source of the organism.
• Glucose after entering a cell can be catabolized either aerobically (in the presence of
O2), where molecular oxygen serves as the final electron acceptor (oxidative
pathway), or anaerobically (in absence of O2) in which inorganic ions can serve as
the final electron acceptor (fermentative pathway).
• The metabolic end products of a carbohydrate fermentation can either be organic
acids (lactic, formic, acetic acid) or organic acid and gas (hydrogen or carbon
dioxide).
• Fermentative degradation of the carbohydrates (monosaccharide, disaccharide, and
polysaccharide) by microorganisms under the anaerobic condition is carried out in
the fermentation tube, which comprises of Durham tube for the detection of the gas
production.
• A fermentation medium is composed of a basal medium containing a specific
carbohydrate (glucose, sucrose, or cellulose) along with a pH indicator (phenol red,
Andrade’s indicator, or bromocresol).
 • When the organism ferments carbohydrates, organic acid products (Lactic acid, Colonial Appearance on Grayish white, small, Grayish white, small,
formic acid, or acetic acid) are obtained which turns the medium into yellow color CA circular, moist smooth. circular, moist, smooth.
with a reduction in the pH (acidic-below pH of 6.8). Growth on NA No Growth No Growth
• The change in the pH indicator in the fermentation tube and the gas production in Oxidase test (+) (+)
the Durham tube is indicative of the metabolic reaction with the production of acid Glucose Acid Acid
end product and gas. Maltose Acid (-)
• Color change only occurs and is visible when a sufficient amount of acid is Sucrose (-) (-)
produced, as bacteria may utilize the peptone producing alkaline by-products. Lactose (-) (-)
• The degradation of peptones in the broth may result in the production of alkaline end
products, which will change the broth color to pink often at the top of the tube.

Procedure
Results and Interpretation 1. Examine the cultures of Mycobacterium tuberculosis demonstrated, the uninoculated
Observations Result Interpretation medium of choice and the microscopic morphology in sputum smears.
The Medium changes to Acid Organism ferments the given 2. Examine the demonstrated smears of skin scraping from patients with lepromatous
Yellow Color Production carbohydrate and produces organic acids leprosy.
thereby reducing the pH of the medium 3. Draw your observations in the worksheet provided for this exercise.
into acidic condition. Mycobacterium leprae
The medium changes to Acid and Gas Organism ferments the given 1. They are acid fast organism and also can be considered as gram +ve bacteria.
yellow and production of Production Carbohydrate and produces organic acids 2. Its cells contain peptidoglycan and stain gram-positive, but most of its cell wall is
gas formation in the durhan and gas. Gas production is detected by comprised of unique types of lipids.
tube. the presence of small bubbles in the 3. Due to thick waxy coating, they stains with carbol fuchsin.
inverted Durham tubes. 4. They are also known as “Hansen’s Bacillus Spirilly”.
No change in color (retains Absence of The organism cannot utilize the 5. They have “Packets of Cigarettes” appearance.
red color) fermentation carbohydrate but the organism continues 6. They have parallel sides with rounded ends.
to grow in the medium using other 7. They are 1-8 µm in length and 0.2-0.5 µm in diameter.
energy sources in the medium. 8. They are non-sporing, non-capsulated and non-motile bacteria.
Limitations 9. They divided by binary fission.
1. Reading after 24 hours may not be reliable if no acid is produced. 10. They appears in group of bacilli side by side.
2. No color change or a result indicating alkalinity may occur if the organism 11. They are slender, slightly curved or straight rods.
deaminates the peptone, masking the carbohydrate fermentation evidence. 12. They appears in clumps and rounded masses.

Basis Neisseria sicca Branhamella catarrhalis

Gram stain Rxn and Gram (-) diplococci, Gram (-) diplococci, Coffee
Bacteriological studies of water have been directed for the most part towards it’s
Microscopic morphology coffee bean shaped. bean shaped.
sanitary aspects because of public health significance of waterborne diseases and the desire to
Colonial Appearance on Small, translucent, raised, Small, translucent, raised,
control it’s spread.
CA moist, grayish white, moist, grayish white,
mucoid. mucoid.
The single best criterion by which the sanitary quality of water may be evaluated is the kind and
Growth on NA numbers of bacteria that are present.
Presence of Growth Presence of Growth
1. Enteric pathogens are unusually outnumbered by the non-pathogenic coliforms
Oxidase test (+) (+) 2. Pathogens often die out more quickly
Glucose Acid (-) 3. Large volumes of water samples are required for isolation
Maltose Acid (-) 4. Isolation and identification of these pathogens would require more complicated time
Sucrose Acid (-) consuming procedures, often these procedures are not always successful and a
Lactose (-) (-) negative finding is of doubtful value
Summary of Results
It is legitimate to assume that when water is polluted with human fecal material, it probably
Basis N. meningitidis N. gonorrhoeae contains bacteria which cause enteric infection and since it is impractical to isolate these
Gram stain Rxn and Gram (-) Diplococci, Gram (-) diplococci, Kidney pathogenic bacteria as a routine measure, some microbial indicator of fecal contamination will
Microscopic morphology Kidney bean shaped. bean shaped. serve equally well as a criterion of the sanitary quality of a water supply.
 • Low temperature are not conducive to rapid growth and tend to keep the numbers of
bacteria down, a factor that favors the survival of pathogens.
Bacteria Native to Natural Waters • Higher temperatures result in an increase of bacterial numbers in the presence of
Bacteria whose natural habitat is water are not well known, partly due to the fact that sufficient organic matter, but if the supply of nutrients is not great, after a preliminary
many of them are difficult to grow on laboratory media. increase during which food supply is exhausted the numbers fall below the initial level.
The group includes the following. • A great majority of bacteria in water are killed by freezing, 90% die within a few hours.
1. Higher bacteria – sulfur bacteria, iron bacteria
2. Appendaged bacteria attached to some inanimate objects Bacteriological Analysis of Water
3. Large spiral forms about 20-30u in length A. Microbial Indicators
4. A variety of bacilli Certain bacteria may be used as indicators of water quality provided their presence in water
a) Pigmented form – Serratia marcescens, Chromobacterium violaceum. is solely the consequence of faecal pollution.
b) Nonpigmented forms – Pseudomonas fluorescens, thermophiles, aerobic and Ideally the bacteriological indicators should:
anaerobic sporeformers. 1. be readily distinguished from native water bacteria
5. Cocci forms: 2. survive as long as or preferably longer than the pathogenic bacteria that may
a) Pigmented – Sarcina lutea accompany it
b) Nonpigmented These include:
6. Nitrogen fixing bacteria 1. Coliform bacilli – present in greatest number
7. Nitrifying bacteria • Since some for the coliforms are found in the environment or occur free in nature, it
Bacterial Contamination of Natural Waters is important to determine whether the coliform present in water sample is of fecal
A. Bacteria from air and soil origin
The number of bacteria in the air bears a close relation to the amount of larger suspended • Faecal coliforms present ferment lactose with acid and gas when incubated at 44C in
particles of dust and which find their way into natural water through settling out or are swept 24 hours (Eijkmann test)
down by the rain. Soil contain tremendous numbers of bacteria most of which are found in the 2. Enterococci (Streptococcus faecalis)
upper 6 inches of soil. • persists longer than pathogenic enteric bacteria but as long as coliforms
A great majority of these are native to the soil, the others are present as contamination coming
• they are usually found in considerably smaller numbers than coliforms
from:
3. Clostridium perfringens
1. flesh of animals and humans who have died of infectious diseases
• Also fewer than coliforms
2. excreta of humans and animals
• it’s disadvantage is that it’s spores remain viable over long periods in contrast to
B. Direct contamination of water by human and animal excreta
coliforms and hence doesn’t permit differentiation of old and recent pollution
Normal intestinal flora which may be found in water:
1. Large numbers of coliform bacilli
In the Philippines and United States of America, the faecal indicator used are the coliforms
2. Clostridium perfringens
whereas in Europe, the faecal indicators used are Streptococcus faecalis and Clostridium
3. Alpha-hemolytic faecal streptococci or enterococci
perfringens.
Pathogenic organisms shed from feces of infected persons and carriers include:
B. Methods of Bacteriologic Analysis of Water Sample
1. Bacterial enteric pathogens: Salmonella, Shigella, Yersinia, Vibrio, Campylobacter,
Enteropathogenic Escherichia coli, Aeromonas and Plesiomonas 1. Multiple Fermentation Tube Technique
2. Parasites: Entamoeba, Giardia, Cryptosporidium, Ascaris, etc. • The standard procedure has been prepared jointly by the American Public Health
3. Enteroviruses: Rotavirus, Norwalk agent, Polio virus, Coxsackie virus, Hepatitis A virus, Association and the American Waterworks Association and is revised at frequent
etc. intervals.
Factors Influencing the Kinds and Numbers of Bacteria • It consists of 3 parts:
A. Type of Water a) Presumptive Test - uses Lactose Broth or Lauryl Tryptose Broth (LTB)
1. Surface water: streams, lakes, shallow wells. b) Confirmed Test - uses Brilliant Green Lactose Bile(BGLB) broth, EC medium
• Opportunities for contamination are great and as might be expected, many bacteria are c) Completed Test - uses Eosin Methylene Blue (EMB) agar, Lactose Broth or Lauryl
present. Tryptose Broth, Nutrient Agar slant for Gram stained smears
2. Water from deep driven wells: water has undergone effective filtration in order to reach • Most Probable Number (MPN) is based on the number of positive BGLB tubes.
the surface strata. • EC Medium – differentiates the presence of non-faecal coliforms and faecal
• It is not subject to an extensive contamination hence this contain only a few bacteria. coliforms.
B. Amount of organic matter present: 2. Membrane Filter Techniique
• In general, the more nutrient there is present in the form of organic matter, the greater • The microbial indicator of water contamination in this procedure is usually the
is the number of bacteria. coliform bacilli, however, enterococci may also be used.
C. Temperature • The test consist essentially of the following:
 a) Filtration of water sample by negative pressure through a cellulose membrane of o An arbitrary limit of 48 hours for observation doubtlessly excludes from
sufficiently small pore to retain the bacteria. considering occasional member of coliform group that form gas slowly and
b) Removal of membrane filter and transfer to an absorbent pad containing differential generally are of limited sanitary significance.
liquid medium such as EMB or Endo medium if coliforms are used as indicator • Submit all primary fermentation tubes showing any amount of gas within 24 hours of
organisms. incubation to the confirmed test.
c) after incubation, the bacterial colonies grow on the surface of the membrane filter o if active fermentation appears in the primary fermentation tubes earlier than 24
and may then be counted. hours, preferable transfer to the confirmatory medium without waiting for full 24
Purification Water Supplies hours.
1. Mechanical methods: o If additional primary fermentation tubes show gas production at the end of 48 hours,
a) storage submit these to the confirmed test.
b) filtration Confirmed Test Day 2
• slow sand filtration 1. Carefully shake each (+) tube in the presumptive test.
• coagulation and rapid sand filtration 2. With the sterile loop, transfer 1 loopful of the culture to BLGB and EC tubes.
2. Chemical methods: 3. Gently agitate the tubes to mix the inoculum and incubate the BGLB tubes at 37C
• large scale - hypochlorite and liquid chlorine while the EC tubes at 44C in the water bath for 24 hours.
• small scale - hypochlorite, UV light, iodine 4. After 24 hours incubation, examine for gas production.
3. Boiling for 5 minutes will kill enteric pathogens 5. After 48 hours incubation, re-examine the BGLB tubes. Record the presence of
absence of gas in the tubes.
WATER BACTERIOLOGY PROPER 6. Discard tubes without gas and keep tubes with gas for the complete test.
Materials 7. Determine the Most Probably Number (MPN) based on the positive BGLB tubes
1. Water samples at least 100 ml in a sterile 200 ml container using the table on MPN index. This is usually the basis for calculations of the MPN
2. (3) Lauryl Tryptose Broth tubes double strength with fermentation tubes coliform bacteria present in the water sample because of the false positive reaction
3. (6) Lauryl Tryptose Broth tubes single strength with fermentation tubes. that can occur in the presumptive test. BGLG is the more selective medium.
4. Brilliant green lactose broth(BGLB) with fermentation tubes Interpretation of Confirmed Test
5. EC Broth with fermentation tubes • (+) Positive Confirmatory test – formation of gas in inverted vial of BGLB
6. Nutrient agar (NA) slant fermentation tube at any time within 48 hours constitute a positive confirmed test.
7. Eosin Methylene Blue(EMB) agar o (+) EC Positive Reaction – gas production in the EC fermentation tube within 24
8. 1-10 ml pipette; 1-ml pipette hours or less is considered a positive reaction indicating fecal origin. (note
9. Inoculating wire loop turbidity)
10. Alcohol lamp ▪ Positive for the presence of fecal coliforms, with gas production.
Procedure o (-) EC Negative Reaction – failure to produce gas (growth sometimes occur)
Presumptive Day 1 constitute a negative reaction indicating the source other than the intestinal tract of
1. Label 9 Tubes of LTB warm blooded animals. (note turbidity)
• 1-3 (3) – LTB 10mL ▪ Negative gas production, presence of non-fecal coliforms.
• 4-6 (3) – LTB 1mL • The Most Probably Number (MPN) of coliform organisms present can be determined
• 7-9 (3) – LTB 0.1mL based on the number of positive tubes obtained in the confirmatory test.
2. Shake the bottle with the water and inoculate all 9 LTB tubes using a sterile pipette. o Class I – regarded as highly satisfactory and contains < 1 coliform/100mL.
3. Mix all the tubes at least 20x o Class II – regarded as satisfactory and contains 1-2 coiforms/100mL.
4. Incubate at 37C for 24 hours. o Class III – regarded as suspicious and contains 3-10 coliforms/100mL.
5. After 24 hours, observe for the presence of gas in the inverted vials. o Class IV – regarded as unsatisfactory and contains > 10 coliforms/100mL.
• If no gas formed, reincubate for another 24 hours and re-examine. Completed Test
6. Observe for the presence of gas in the inverted vials. 1. Streak an EMB Plate from each tube of BGLB showing gas. Streak plates in such a
7. Submit all (+) tubes to confirmed test. manner to ensure the presence of discrete colonies separated by at least 0.5cm.
Presumptive Test Interpretation 2. Incubate plates at 37C for 24 hours.
• (+) Positive Presumptive Test – formation of at least 10% gas in the inner fermentation 3. From each plate:
tubes or vials within 48 hours constitute a positive presumptive test. (note turbidity) o Pick 1 or more typical well isolated coliform (colored) colonies
o If gas is formed as a result of fermentation the broth medium will become cloudy. 4. Or if no typical colony is present
o Active fermentation may be shown by the continued presence of small bubbles of o Pick 2 or more colonies considered most likely to consist of organisms of the
gas throughout the medium outside the inner vial when tube is shaken gently. coliform group and transfer growth from each isolated colony to Lauryl Tryptose
Broth Fermentation Tube and to Nutrient Agar Slant.
• (-) Negative Presumptive Test – absence of gas formation at the end of 48 hours of
incubation.
 5. Incubate LTB Tubes at 37C for 24 hours and observe for gas production. If no gas
production, reincubate for another 24 hours.
6. Make gram stained smears of colonies from Nutrient Agar Slant and look for Gram
(-) nonsporing coccobacilli.
Completed Test Interpretation
• Colonies developing on EMB are called: (after 24 hours incubation)
o Typical – dark, flat, ringed with or without metallic sheen.
o Atypical – pink, raised or convex, mucoid opaque)
• (+) Positive Completed Test - Formation of gas in the second tube of LTB within 48
hours and demonstration of Gram (-) non-spore forming rod-shaped bacteria in the
NA slant, demonstrating the presence of a member of the coliform group.
Total Bacterial Count (3M Petrifilm Aqua Heterotrophic Count Plate)
1. Place 3M Petrifilm Aqua Plate on a level surface. With the pipette perpendicular to
the 3M Petrifilm, place 1mL of sample OR hydration diluent onto the center of the
bottom film.
2. Carefully roll top film down so that it contacts the sample or hydration diluent and
then drop the top film.
3. With ridge side down, place spread on top film over inoculum or hydration diluent.
4. Gently apply pressure on spreader to distribute inoculum or hydration diluent over
circular area before gel is formed. (do not twist or slide spreader, LIFT the spreader).
5. Incubate 3M Petrifilm Aqua AQCC Plates in a horizontal position, clear side up, in
stacks on no more than 20 plates at 35 +1C for 24 +2 hours or 36 +1 C for 24 +2
Hours.
6. 3M Petrifilm Aqua AQCC Plates can be counted on standard colony counter or other
illuminated magnifier.
Colony Counting
• Select the plate in which the colonies are evenly distributed and containing 30-300
colonies.
• Count all the colonies including those of pinpoint size.
• The use of the Quebec Colony Counter is recommended.
• Calculate the colonies/cu.mm by multiplying the number of colonies in the plate by
the fraction of cu.mm of sample used.
Total Bacteria Count Interpretation
• Analysis consist in counting the organisms present in a given unit volume of water on
a standard agar medium incubated at 37C for 24 hours.
• It is not a true total count for it misses dead bacteria and those that do not grow at 37C
and bacteria that does not form visible colonies within 24 hours under standard
condition.
• For this reason, it should be reported either as the number of number of colonies
developing/mL or simple plate count/mL
 Presumptive Confirmed Test Completed Test
Water
Group MPN Colony Count Remarks
Sample 10mL 1mL 0.1mL BGLB EC EMB LTB NA

Positive
for fecal Typical Positive for
>2,400
Shallow coliforms Greenish Turbid Gram (-) TNTC: > fecal
1 3 3 3 3-3-3 coliforms
Well and Metallic w/gas coccobacilli 500CFU/mL coliforms
/100mL H2O
presence Sheen Class IV
of gas
Negative;
Positive
Typical Positive for
for
Private 9 coliforms Greenish Turbid Gram (-) TNTC fecal
2 3 0 0 2-0-0 Presence
Pump /100mL H2O Metallic w/gas coccobacilli >500CFU/mL coliforms
of non-
Sheen Class III
fecal
coliforms.
Positive
Positive or
for fecal Typical
fecal
Household coliforms 23coliforms Greenish Turbid Gram (-) TNTC; >500
3 3 1 1 3-0-0 coliforms
1 and /100mL H2O metallic w/gas coccobacilli CFU/mL
and Class
presence sheen
IV
of gas
Positive
for Fecal Typical Positive for
Shallow Coliforms 23 coliforms Greenish Turbid Gram (-) TNTC: > fecal
4 3 1 1 3-0-0
Well 2 and /100mL H2O Metallic w/gas coccobacilli 500CFU/mL coliforms
presence Sheen Class IV
of Gas
Positive
for Fecal Typical Positive for
Shallow Coliforms 75coliforms Greenish Turbid Gram (-) TNTC: > fecal
5 3 3 3 3-1-1
Well 3 and /100mL H2O Metallic w/gas coccobacilli 500CFU/mL Coliforms,
presence Sheen Class IV
of Gas
Positive
Typical Positive for
for Fecal Turbid
Private 9 coliforms Greenish Gram (-) TNTC: > Fecal
6 2 1 0 2-0-0 Coliforms with
Pump 2 /100mL H2O metallic coccobacilli 500CFU/mL Coliforms
Presence Gas
sheen Class III
of Gas
Negative;
Positive
Typical Positive for
for
Household 9 coliforms Greenish Turbid Gram (-) TNTC: > fecal
7 3 0 0 2-0-0 Presence
2 /100mL H2O Metallic w/gas coccobacilli 500CFU/mL coliforms
of non-
Sheen Class III
fecal
coliforms.
Positive
for Fecal Typical Positive for
9 coliforms
Shallow Coliforms Greenish Turbid Gram (-) TNTC: > fecal
8 3 3 3 2-0-0 /100mL of
Well 4 and Metallic w/gas coccobacilli 500CFU/mL coliforms
H2O
presence Sheen Class III
of gas
 4. A Quebec colony counter may be used and proceed as in water bacteriology to
Many diseases may be transmitted through milk, among them are: determine the total number of colonies per mL
• Tuberculosis, 5. Report as plate count/mL
• Brucellosis, Procedure: Coliform Count in Milk
• Epidemic sore throat, Medium: Violet Red Bile Agar
• Diphtheria, 1. Transfer 1 mL of milk to a sterile petri dish.
2. To each petri dish with 1mL of milk sample, pour a tube of melted Violet Red Bile
• Typhoid fever,
Agar. Mix and allow to harden and incubate at 37C for 24 hours.
• Paratyphoid fever
3. Count the number of bacterial colonies present on the plate.
• Dysentery. 4. A Quebec colony counter may be used and proceed as in water bacteriology to
Some of them arise from the cow itself, but many are due to the contamination of milk in determine the total number of colonies per mL of milk
subsequent handling by infected individuals or carriers. Fortunately, most of these are destroyed
by Pasteurization The results obtained from microbiological analysis of milk provide useful Group Sample Plate Count/ml Coliform Count/ml Remarks
information reflecting the conditions under which it has been produced and held.
• High Bacterial counts do not necessarily mean that the disease-producing bacteria are 1 Pasteurized Milk No Growth NO Growth Certified (Pasteurized)
present, they do, however, indicate contamination
• They may also indicative of improper cooling and holding temperatures. 2 Raw Milk 1 304 88 Grade C
• Milk grading is largely done with the use of plate count or it’s variation.
Following Classification is adapted in our Public Health System: 3 Pasteurized Milk No Growth No Growth Certified (Pasteurized)
GRADE Plate Count/mL not to Coliform Count/mL not to
exceed exceed 4 Raw Milk 2 112 18 Grade C
Certified (Pasteurized) 500 1
Certified (Raw) 10,000 10 5 Pasteurized Milk No Growth No Growth Certified (Pasteurized)
Grade A (Pasteurized) 30,000 10
Grade B (Pasteurized) 50,000 10 6 Raw Milk 3 2 1 Certified (Pasteurized)
Grade C No Limit No Limit
7 Pasteurized Milk No Growth No Growth Certified (Pasteurized)
• The number of Escherichia coli or coliforms in milk is a rough indication as to how milk
has been handled before it reaches the processing plant.
8 Raw Milk 2 112 18 Grade C
• A large number of coliforms indicate the use of dirty equipment and/or open
contamination of milk.
• This can be easily shown by the use of Violet Red Bile Agar (VRBA) - a selective and .
differential medium –
• The presence of coliforms is demonstrated by the appearance of purplish red colonies
surrounded by precipitated bile.

Materials
1. Milk samples (raw and pasteurized)
2. Tryptone Glucose Extract Agar.
3. Violet Red Bile Agar
4. Sterile 1mL pipet
5. Alcohol Lamp

Procedure: Viable Bacterial Count in Milk

Medium: Tryptone Glucose Extract Agar
1. Transfer 1mL of milk sample to a sterile petri dish.
2. To each petri dish with 1mL milk sample, pour a tube of melted Tryptone Glucose
Extract Agar. Swirl on the desk top to mix. Allow to harden and incubate at 37C for
24 hours.
3. Count the number of bacterial colonies present on the plate.
 Biochemical Tests
MEDIUM READING INTERPRETATION READING INTERPERTATION
TEST DESCRIPTION PROCEDURE REAGENTS
POSITIVE NEGATIVE
INDICTORS
• TSI agar is a differential medium which is used
to determine the ability of the organism to
attack a specific carbohydrate, with or without
the production of gas, along with the
determination of possible hydrogen sulfide Bold = Reading
production Italic - Interpretation
• Recommended for use in the ID of Gram (-)
enteric pathogens in the routine examination of 1. Use a single, well • A/A - Yellow slant, yellow butt
stools isolated colony for ✓Dextrose fermented, lactose and/or sucrose fermented
• Organisms fermenting dextrose only will inoculation • Alk/A - Red slant, yellow butt
originally give an acid (yellow) butt and slant. 2. With a wire needle, ✓ Dextrose fermented, lactose and sucrose not fermented
However due to the small amount of dextrose, it inoculate the TSI by • Alk/Alk or Alk/NC - Red slant, red/orange butt
Triple Sugar Iron is used up as incubation continues. The slant, making a line on the ✓ Dextrose, Lactose & Sucrose are not fermented
Agar Reaction under aerobic conditions, reverse to alkaline slant surface then M: TSI agar
(TSI) (red) in 18-24 hours. stab the butt until a • An H2S producing organism may produce so much black precipitate
• In the butt where anaerobic condition prevails, few mm from the (Ferrous Sulfide) that the acidity in the butt is completely masked
there is no reversion and the acid reaction bottom then streak • However, if H2S is produced, an acid condition does exist in the butt
remains the surface of the even if not observable and should be recorded as such.
• There is 10x more lactose and sucrose present slant
than dextrose, so organisms fermenting either 3. Incubate at 37°C for • Gas production is indicated by
or both of these sugars do not use them up, 24 hrs ✓ Splitting of the medium
except after a very prolonged incubation. With ✓ Single gas bubble
prolonged incubation, 48-72 hours, lactose or ✓ Complete displacement of medium from bottom of tube
sucrose may also be used up and formerly acid ✓ Slight indentation of the medium from side of tube
reactions may reverse to alkaline
• Incubation time is crtical; 18 hrs incubation is
recommended to obtain typical reactions
• LIA is for determining whether the organisms
can decarboxylate or deaminate lysine
• Lysine decarboxylase production is indicated by
the reaction in the butt. Enteric bacilli will
produce acid in an initial fermentation of • P/P - Purple slant, purple butt
dextrose with a color change of the agar to ✓Absence of lysine deaminase, presence of lysine decarboxylase
yellow. Those cultures which produce lysine 1. Stab the butt twice all ✓Lysine was not deaminated but was decarboxylated
Lysine
decarboxylase form cadaverine and the agar the way down then ✓Cadaverine was produced
Decarboxylation M: Lysine Iron Agar
reverts to the purple alkaline color. The lysine streak the slant • P/Y - Purple slant, yellow butt
and Deamination I: Bromcresol Purple
decarboxylase reaction can only be detected surface ✓ Absence of both lysine deaminase and decarboxylase
(LIA)
with organisms which ferment dextrose. 2. Incubate at 37°C for • R/Y - Red slant, yellow butt
Nonfermenters cannot be tested by this method 18-24 hrs ✓ Presence of lysine deaminase, absence of lysine decarboxylase
LDC cadaverine
purple
-
-

• Lysine deaminase (formed only by Proteus and ✓ alpha-ketocarboxylix acid was produced
=

a Keto carboxylic Providencia with a few

=
L DM
-

acid red
• exceptions is shown by the formation of a red
=

slant, a yellow butt is also produced by these

organisms, as Proteus and Providencia are

Compiled by JanMomo
 lysine decarboxylase negative. Occasional
cultures of Morganella morganii do not give this
red reaction
• It is essential that the medium be tubed in at
least a depth of 2 inches to provide the
Turbid and Presence of Absence of
anaerobic conditions necessary for the detection (-) Non Motile
Fuzzy flagella Flagella
of ornithine decarboxylase activity
• Positive/Motile - Motile organisms migrate 1. Stab the medium
Motility and from the stab line and diffuse into the medium once with a straight
causing haziness; may exhibit fuzzy streaks of
Ornithine growth wire needle to the
M: MIO Purple
Decarboxylation • Negative/Non-Motile - Bacterial Growth bottom of the tube
(MIO) 2. Incubate at 37°C for Throughout
accentuated along stab line; surrounding
24 hrs or 2/3 of the Putrescine was Bottom is Putrescine was
medium remains clear. Stab line is visible.
medium from Produced bright yellow not produced
• OD (-) – Bottom of the medium will be bright
the top is
yellow with a narrow rim of purple at the top.
purple w/ narrow

• OD (+) – Purple color throughout the tube or purple

rim

2/3 of the medium from the top is purple.

• Indole is produced as a putrefactive product
from tryptophan and other amino acids 1. Inoculate 1 Loopful of
containing the indole ring and only under organism into the
aerobic conditions and the absence of sugars tube of the tryptone
Red Ring after
from the medium broth.
M: Tryptone Broth addition of Presence of Absence of
Indole Production • The ability of an organism to give a positive 2. Incubate at 37C for Yellow Ring
R: Kovac’s Reagent Kovac’s Tryptophanase Tryptophanase
indole test depends on whether or not it 24 hours.
Reagent
possesses the enzyme tryptophanase. 3. In the next period,
• Indole (+) – a red ring on top of medium after add 1cc of Kovac’s
addition of Kovac’s Reagent. jpurph.gg if Erlich 's Reagent.
• Indole (-) – yellow ring is observed.

• Citrate was
1. Inoculate the surface utilized
• Simmon’s Citrate Agar (SCA) is used to
of SCA by streaking • Presence of
determine if an organism is capable of utilizing M: Simmon’s Citrate
the loopful of Deep Citratase
Citrate Utilization citrate and ammonium salt as the sole source of Agar Green No Change
organism. Prussian Blue • Sodium
carbon and nitrogen respectively for I: Bromthymol Blue
2. Incubate at 37C for Bicarbonate and
metabolism with resulting alkalinity.
48 hours. Ammonia were
produced.

1. Inoculate a heavy
• Some bacteria posses the ability to split urea • Presence of
loopful of the
into ammonia and carbon dioxide which is urease.
organism from TSI to M: Urea Broth No color Absence of
Urease Test accomplished by enzyme urease. Dark Pink • Ammonia and
urea broth. I: Phenol Red change urease.
• This test is done for rapid identification of Carbon dioxide
2. Incubate at 37C for
proteus organisms. were produced.
24 hours.

Compiled by JanMomo
 1. Streak the slant • Presence of
surface of the phenylalanine
• Phenylalanine is used to determine the ability of phenylalanine agar Green Slant deaminase.
M: Phenylalanine Cannot
Phenylalanine the organism to deaminate phenylalanine to with organism. after addition • Phenylalanine No Color
Agar deaminate
Deamination phenylpyruvic acid by enzymatic activity with 2. Incubate at 37C for of 10% Ferric is deaminated Change
I: Ferric Chloride phenylalanine.
resulting acidity. 24 hours. Chloride • Phenylpyruvic
3. Add 5 drops of Ferric acid was
Chloride. produced.

• Malonate broth is used to determine the

organism’s ability to utilize sodium malonate as
the sole source of carbon with resulting 1. Inoculate Malonate
alkalinity. broth with a heavy
• This medium is prepared with ammonium loopful of overnight • Malonate was
sulfate and sodium malonate as the only source growth from TSI (do Light blue to utilized. No Color
Malonate M: Malonate Broth Does not utilize
of nitrogen and carbon. not use growth from deep Prussian • Sodium Change
Utilization I: Bromthymol blue malonate.
• Organisms which utilizes malonate as a source MacConkey as false blue medium. Hydroxide was (Green)
of energy produce alkaline reaction and a negative may occur.) produced.
change of color medium while those that are not 2. Incubate at 37C for
capable leaves the medium unchanged. 24-48 hours.
• The alkalinity is due to the formation of sodium
hydroxide (NaOH).
1. Streak the slant
surface of nitrate
agar with the
organism.
Red Color • Presence of
2. Incubate at 37C for
• The reduction of nitrates to nitrites or free M: Nitrate Agar after addition Nitratase.
Reduction of 48 hours. No Change in Absence of
nitrogen gas can be accomplished by the enzyme R: Sulfanilic Acid and of sulfanilic • Nitrate was
Nitrates to Nitrite 3. After 48 hours, add 2 Color NItratase.
nitratase in the presence of hydrogen donor. Alpha-napthylamine acid and a- reduced to
drops of sulfanilic
naphtylamine nitrite.
: acid followed by 2
end product drops of alpha-
1 a mi n e
a to a
napthy napthylamine on the
-

1to be n te n e
-
-

p -
su

surface of the agar.

• Organisms ferment carbohydrates in different
ways depending upon the enzymes they possess. 1. Inoculate the
• Pyruvic acid may be considered as the key organism into the
compound formed during the process of solid sugar media in
• A/A(G) – Yellow with/without gas production.
fermentation since it is at that the pathways of butt preparation by
Fermentation of I: Andrade’s ✓ Sugar was fermented, aerogenic or anaerogenic.
carbohydrate metabolism diverge with different stabbing up to a few
Carbohydrates Indicator ✓ Sugars – Lactose, Glucose, Rhamnose, Arabinose, Inosite.
species of bacteria. mm from the bottom
• Negative – No Change in color of medium or pink.
• Pyruvic acid may be converted into a variety of with a needle.
end products. 2. Incubate at 37C for
• The test differentiates the fermentative capacity 24 hours.
of pathogens.
Pigment For Pseudomonas For Serratia
Production ✓ PAP – Pyocyanin is produced. ✓ PAP – Prodigiosin is produced.
✓ PAF – Pyoverdin or Fluorescein is produced.

Compiled by JanMomo
 Biochemical Tests (Grouped by TSI Reaction)
TSI MIO CARBOHYDRATES PIGMENTS
ORGANISM LIA IND CIT URE PAD MAL NIT
SLANT BUTT H2S OD MOT Lac Glu Ara Rha Ino PAP PAF
A/AG
Escherichia
A AG - P/P - (+) M(-) + - - - - + A A A A - - -
coli
Enterobacter
A AG - P/P + M - + - - + (-) + A A A A A - -
aerogenes
Klebsiellia
A AG - P/P - NM - + + - + + A A A A A - -
pneumoniae
Enterobacter
A AG - P/Y + M - + - (+) - + (-) + A A A A A - -
cloacae
ALK/A or ALK/AG
Serratia ALK +
A (G) - P/P + M - + - (+) - - + A A - - A -
marcescens (A) (prodigiosin)
Providencia ALK
A - R/Y - M + + + + - + - A - A A - -
rettgeri (A)
Morganella
ALK A (G) - R/Y + M + - + + - + - A -- - - - -
morganii
Citrobacter A
AG + (-) P/Y + (-) M - + - - - + A A A A - - -
fruendii (ALK)
Salmonella
ALK A (G) - P/Y + M - - - - - + - A A A - - -
paratyphi-A
Shigella
ALK A - P/Y + NM - - - - - + - A A A - - -
Sonnei
-
Shigella
ALK A - P/Y - NM - (+) - - - - + - A - - - - -
dysenteriae
Shigella
ALK A - P/Y - NM + (-) - - - - + - A - A A - -
flexneri
ALK/A or ALK/AG with H2S
Proteus ALK
A (G) + R/Y - M + - (+) + + - + - A - - - - -
vulgaris (A)
Proteus ALK
A (G) + R/Y + M - + (-) + + _ + - A - - - - -
mirablis (A)
Salmonella
ALK AG + (-) P/P + M - - (+) - - - + - A - A - - -
choleraesuis
Salmonella
ALK A (G) + P/P + M - + - - - + - AG AG A A - -
enteritidis
Salmonella
ALK A (G) + P/P + M - + - - - + - A A A - - -
typhimurium
Salmonella
ALK A + P/P - M - - - - - + - A - - - - -
typhi
ALK/ALK
Pseudomonas + +
ALK ALK - P/P + M - + - - + + - - - - -
aeruginosa (pyocyanin) (pyoverdine)
Alcaligenes
ALK ALK - P/P + M - + - - + + - - - - - - -
faecaclis
NOTE: readings outside of the parenthesis are the results from the ppt given. Answers inside ( ) are from the manual.
corrections are much appreciated.
Compiled by JanMomo
 Media for Isolation (based on their Medium of Choice)
MEDIUM TYPE OF MEDIUM DESCRIPTION BACTERIA APPEARANCE
• Sugars
o Lactose
o Sucrose Small sized, circular, blue-black colonies with dark
Escherichia coli
• Indicators centers and green metallic sheen.
o Eosin Y Proteus vulgaris Small, translucent, colorless swarming growth.
o Methylene Blue Klebsiella pneumoniae Large, pink, mucoid, capsulated colonies.
• Inhibitors Large, pink with purple center, mucoid colonies with
Enterobacter aerogenes
o Eosin Y fish-eye appearance.
o Methylene Blue Enterobacter cloacae Fish-eye colonies
Eosin Methylene Differential Mildly
• Lactose Fermenters Citrobacter freundii Round, greenish metallic sheen
Blue Agar (EMB) Selective
o Colored colonies. Proteus mirabilis Small, circular, translucent, colorless
o I.E. pink or with greenish sheen. Morganella morganii Translucent, pinpoint, colorless
• Non-Lactose Fermenters Providencia rettgeri Small, mucoid
o Colorless Alcaligenes faecalis Small, colorless translucent colonies
o Takes up the color of the medium. Large, white to pinkish colored colonies with fish-eye
Serratia marcescens
• contain carbohydrates and pH indicators with other chemicals appearance.
that are inhibitory to Gram (+) to bacteria Pseudomonas aeruginosa Small to medium sized, colorless translucent colonies
• it is differential because it can differentiate lactose fermenters
from nonlactose fermenters
• Sugars
o Lactose
• Indicators
o Neutral Red
o Ferric Citrate
o Thiosulfate
Shigella dysenteriae Transparent, colorless
• Inhibitors
o Sodium Citrate Shigella flexneri Translucent, colorless
o Brilliant Green Shigella sonnei Small, pink, entire
Salmonella Differential o Bile Salts
Shigella Agar Moderately • Lactose Fermenters Salmonella paratyphi A Medium, pink, raised entire, opaque, mucoid
(SSA) Selective o Pink Salmonella choleraesuis Small to Medium-sized, colorless colonies, some with
• Non-Lactose Fermenters black centers
o Colorless Salmonella enteritidis Medium-sized, colorless colonies with black centers.
o Takes up the color of the medium. Salmonella typhimurium Translucent, colorless
• designed for the isolation of Salmonella and Shigella which are
overt pathogens
• contain carbohydrates and pH indicators with other chemicals
that are inhibitory to Gram (+) to bacteria and most of the
coliforms
• •SSA: produce colorless colonies
• Sugars: Glucose
Highly Selective • Indicators: Ferrous Sulfate, Bismuth Sulfite
Bismuth Sulfite Salmonella typhi Jet-black colonies
for Salmonella • Inhibitors: Brilliant Green.
Agar (BSA)
typhi • Lactose Fermenters - Inhibited
• Non-Lactose Fermenters - Black colonies with metallic sheen.
*morphological descriptions are from the senior notes.
Compiled by JanMomo