LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Laboratory # 7
Identification and Differentiation of Haemophilus species
Skills= 10 Points
Objectives:
At the completion of this laboratory, the student will be able to:
1. Inoculate, observe, and describe the cultural characteristics of the following
organisms on chocolate agar:
a. Haemophilus influenzae
2. Accurately perform and interpret the following tests:
a. X and V factor using Haemophilus Quad plate
b. Satellite phenomenon
c. Beta lactamase
3. Discuss the satellite phenomenon.
4. Explain the gram stain morphology of Haemophilus.
5. Explain the principle of the Staphylococcus streak method and its interpretation.
Materials:
Chocolate agar cultures:
Haemophilus influenzae
2 Glass Microscope slides
Microscope
Immersion Oil
Gram stain reagents
1 Blood agar plate (BAP)
3 sterile swabs
1 Beta-Lactamse/Cefinase disc
Trypticase Soy Broth OR Di H20
1 Haemophilus Quad plates
Culture of Staphylococcus aureus
1 RapID NH System Panel
Rap ID Nitrate A Reagent
Rap ID Nitrate B Reagent
Rap ID Spot Indole Reagent
References:
1. Mahon and Manuselis, Textbook of Diagnostic Microbiology, Fourth Edition,
Chapter 18
2. Remel NH Panel product insert
3. Rowland, Walsh, Teel, Carnahan, Pathogenic and ClinicalMicrobiology- A Lab
Manual, Chapter 6
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Principles:
As implied by the genus name Haemophilus (blood loving), this group of bacteria
requires certain factors derived from blood before any of the species will grow on
laboratory culture media. Some species require Factor X (hemin), a heat-stable, iron
protoporphyrin derivative of hemoglobin. Others require nicotinamide adenine
dinucleotide (NAD), also known as Factor V, and some require both growth factors.
Factor X can be derived from a peptic digestion of blood or from heat-disrupted
erythrocytes as used in chocolate agar.
Factor V is heat-labile. It can be derived from extracts of yeasts or potatoes and is
produced by certain bacteria such as Staphylococcus aureus. Factor V is not found in
regular blood agar because blood contains NADase, which destroys Factor V or NAD.
Most species of Haemophilus require an increased CO2 environment (candle jar) for
good growth. The genus Haemophilus consists of small, nonmotile, pleomorphic bacilli
that can range in morphology from coccobacilli to filamentous rods. Members of the
genus Haemophilus constitute part of the normal upper respiratory tract. H.influenzae
is a major cause of invasive disease such as septicemia, meningitis and epiglottitis,
especially in young children. Strains of H.influenzae that cause invasive disease almost
always possess a type b polysaccharide capsule, which is the main virulence factor for
this organism.
Procedure:
1. Observe the appearance of the colonies on chocolate agar of H.influenzae.
Describe the characteristics on the report form at the end of this exercise.
2. Perform a gram stain on Haemophilus influenzae and record results on the
report form at the end of this laboratory exercise.
3. X and V Factor Requirements
a. Staphylococcus Streak Technique
1) Many microorganisms, including Staphylococci, Neisseria, and
certain species of yeast, can synthesize NAD (Factor V). When
these organisms are present in mixed cultures, species of
Haemophilus requiring Factor V may appear as small dew-drop
colonies within the zones of NAD production, around colonies of
the other bacteria, a phenomenon known as satellitism.
2) Take a colony of Haemophilus influenzae and streak as a lawn
on a blood agar plate.
3) Using an inoculating loop, make a circle (see diagram) of S.
aureus in the area where the specimen has been inoculated.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
The staph streak is made in the zone of secondary inoculation.
4) After 18 to 24 hours of incubation at 35° to 37°C in CO2
incubation, tiny, moist, dew-drop colonies of Haemophilus may
be observed within the hemolytic zone adjacent to the
staphylococcus colonies.
5) Record results by notating the presence or absence of satelliting
colonies.
4. Haemphilus Quad Plates
a. The Haemophilus identification plate is used to determine the hemolytic
properties and requirement for X and V factors for particular
Haemophilus species. The plate consists of four quadrants:
Quadrant I: BHIA and hemin (X)
Quadrant II: BHIA and Isovitale (V)
Quadrant III: BHIA with hemin (X) and isovatale (V)
Quadrant IV: Horse blood agar (X) with NAD (V)
Presence or absence of growth in each quadrant and hemolytic
properties are used to speciate Haemophilus. Hemolysis is observed in
quadrant IV.
b. Pick several colonies of suspected organisms with a sterile inoculating
needle or cotton swab. Suspend organism in 5 mls of trypticase soy broth
or Di H2O (NO BLOOD BANK SALINE!) until a turbidity of 0.5 McFarland is
obtained.
c. Streak one loopful of this suspension on each quadrant of the plate.
Streak the entire quadrant. Stab blood agar quadrant. Sterilize loop
between quadrants.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
d. Incubate plate in 3% to 10% CO2 at 35oC for 18 to 24 hours.
e. Interpret plate for visible growth and hemolysis. The organism should
grow in quadrants III and IV. Read quadrant IV for hemolysis. Growth in
quadrants II, III, and IV indicates requirement for factor V. Growth in
quadrants I, III, and IV indicates requirement for factor X. Growth only in
quadrants III and IV indicates requirement for both factors V and X.
HAEMOPHILUS QUAD PLATE
GROWTH IN QUADRANTS
II (V)
III (XV)
IV(HEMOLYSIS)
HAEMOPHILUS SPECIES
I (X)
Haemophilus influenzaee
-
-
+
-
-
+
+
-
-
-
+
+
-
+
+
+
Haemophilus
parainfluenzaee
Haemophilus
haemolyticus
Haemophilus
parahaemolyticus
5. Beta-Lactamase/ Cefinase Testing
Perform a beta-lactamase on H. influenzaee and record results as positive or negative.
Refer to lab #6 for procedure if needed.
6. Remel NHI Panel
Principle: This procedure is a qualitative micromethod that utilizes conventional and
chromogenic substrates to identify medically important species of Neisseria and
Hemophilus. The NH system consists of several reaction cavities molded into the
periphery of a plastic disposable tray. The reaction cavities contain dehydrated
reactants and the tray allows for the simultaneous inoculation of these reaction wells.
After incubation of the panel, each test cavity is examined for test reactivity by notating
the development of a color. In some of the wells, reagents must be added to the test
cavities to provide a color change. The resulting pattern of positive and negative test
scores is used as the basis of identification of the test isolate by comparison of test
results to certain reactivity patterns stored in a database.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Procedure:
1. For this exercise, we will utilize H. influenzae as the test organism. Notate the
results of your gram stain on the RapID NH result sheet. Perform an oxidase on
the organism.
2. It is preferable to use an inoculum from a Chocolate plate or a Thayer-Martin
Agar. The inoculum should originate from a culture approximately 18-24 hours
old. Using a cotton swab or inoculating loop, suspend sufficient growth from the
agar plate into 1 mL of saline to achieve a turbidity approximately equal to #3
McFarland standard.
3. Peel back the lid of the panel over the inoculation port by pulling the tab marked
“Peel to Inoculate” up and to the left.
4. Using a pipette, transfer the entire contents of the inoculation fluid tube into the
upper right hand corner of the panel. Reseal the inoculation port by pressing the
peel-back tab into place.
5. After adding the test suspension, and while keeping the panel of a level surface,
tilt the panel away from the test cavities at approximately a 45- degree angle.
6. While tilted back, gently rock the panel from side to side to evenly distribute the
inoculum along the rear baffles
7. While maintaining a level, horizontal position, slowly tilt the panel forward
toward the reaction cavities until the inoculum flows along the baffles into the
reaction cavities.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
8. Return the panel to a level position. If necessary, gently tap the panel on the
bench to remove any air trapped in the cavities.
9. Incubate the panel at 35-37o C in a non-CO2 incubator for 4 hours.
10. Scoring of the panel:
a. While firmly holding the RapID™ NH panel on the benchtop, peel back the lid
over the reaction cavities by pulling the lower right hand tab up and to the
left.
b. Without the addition of any reagents, read and score cavities 1 (PRO) through
10 (URE) using the interpretation guide that follows. Record the results in the
unshaded boxes on the report form.
i. Cavities 1-10 contain the following tests:
Test: PRO GGT ONPG GLU SUC EST RES PO4 ORN URE
Cavity: 1
2
3
4 5
6 7 8
9
10
ii. Test Interpretation:
POSITIVE
Yellow
NEGATIVE
Clear or Tan
Yellow, Gold, or Yellow-Orange
Red, Red-Orange, or Orange
Pink
Purple, Blue, or Violet
Cavity 8 (PO4):
Yellow
Clear, Tan, Straw, or very pale
yellow
Cavity 9 (ORN):
Cavity 10 (URE):
Red, Violet, or Purple
Red, Violet, or Purple
Yellow or Orange
Yellow or Orange
Cavities 1-3:
(PRO,GGT,ONPG)
Cavities 4-6:
(GLU,SUC,EST)
Cavity 7 (RES):
iii. Cavities 8 through 10 are bifunctional, containing two separate
tests in the same cavity. After reading the results for cavities 1-10,
add the following reagents to the cavities indicated.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
1. Add 2 drops of RapID™ Nitrate A Reagent to cavity 9 (NO3).
2. Add 2 drops of RapID™ Nitrate B Reagent to cavity 9 (NO3).
3. Add 2 drops of RapID™ Spot Indole Reagent to cavity 10
(IND).
NOTE: Only RapID™ Spot Indole Reagent should be used.
Kovac’s or Ehrlich’s Reagent will not provide satisfactory
results.
Allow at least 1 minute but no more than 5 minutes for color
development. Read cavities 9-10 and record the scores in
the shaded boxes on the report form.
iv.
Cavities 9 and 10 contain the following tests after reagent addition:
Test:
NO3 IND
Cavity: 9 10
Cavity 9 (NO3):
Cavity 10 (IND):
Test interpretation after reagent addition:
POSITIVE
NEGATIVE
Red or Orange
Yellow
Brown or Black
Orange or Red
v. If the PRO test (cavity 1) is the only POSITIVE reaction, and the
remaining tests are NEGATIVE, then a NITRITE test should be
performed by adding 2 drops of RapID™ Nitrate A Reagent and 2
drops of RapID Nitrate B Reagent to cavity 8 (NO2). Allow a full 5
minutes for color development before interpreting Nitrite (NO2)
reaction.
Cavity 8 contains the following test after reagent addition:
Test: NO2
Cavity: 8
Cavity 8 (NO2):
Test interpretation after reagent addition:
POSITIVE
NEGATIVE
Clear, Tan, or Straw
Pink or Red
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
vi.
Reference the microcode obtained on the report form in the
RapID
NH Compendium found on the lab computer.
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Lab #7: Identification and Differentiation of Haemophilus sp.
NHI Report Sheet
Points= 2
Student name: ________________
MLAB 2534- Laboratory 7
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Name:____________________
Date:_____________________
Points= 8
Lab #7: Identification and Differentiation of Haemophilus sp.
Report Sheet
Quad Plate
Organism
Colony
Morphology
Gram Stain
Staph Streak
Technique
Q:
I
Q:
II
Q:
III
Haemophilus
Influenzaee
MLAB 2534- Laboratory 7
Q:
IV
β lactamase
LABORATORY #7
Identification and Differentiation of Haemophilus sp.
Name :_______________________
Date :________________________
Lab #7 : Haemophilus
Study Quesions
Points= 5
1. What atmosphere and medium should be used for the culture of H. influenzaee ?
2. What is the satellite phenomenon ?
3. CSF Culture
Growth on BAP : no growth
Growth on CHOC : many tan colonies
Gram stain : GNCB
X&V Plate :
Quad I : X
Quad II : V
Quad III : X&V
Quad IV : Horse Blood
No hemolysis
No growth
Quad IV
Quad I
Quad III
Quad II
No growth
Growth
Identification :_________________________________
Diseases caused :__________________________________
MLAB 2534- Laboratory 7