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السبت، 27 أكتوبر 2012

Rapid flickering Chicarda tips to calm rapid heartbeat

Rapid flickering Chicarda tips to calm rapid heartbeat
This situation comes suddenly, and you do not understand something about the condition of your heart and all of a sudden become does go fast! The increasing number of pulses of 72 beats per minute to 120-180-200 in seconds pulse, breathing and may increase as well, and become a state of nausea with panic, and begin to sweat.The doctor says: You infected does go fast, and specifically, that you have accelerated heart paroxysmal, no doubt that once injury in this case for the first time you make a full examination, have you and your doctor to exclude injury quickly ventricle (a type of rapid heartbeat life threatening) and all diseases organic heart like thyroid disorders and pulmonary insufficiency. . . Etc.. That made you feel reassured.However, often come out atrium - a room in the heart receives blood from the veins and pumped into the ventricles - a bit of control, The atrium has a regular rhythm, but this rhythm can be faster than the normal three times.I know that the rapid flickering indicates increased heart rate faster than 100 beats per minute.
There are ways and means to reduce the heart rate, and below you will find methods to help you overcome the heart, in addition to tips on lifestyle for the prevention of heart:You calm:See this situation as a red flashing signal and says: stop what works, step back and tried to calm down. The rest is the best way to stop any bout.Vagal maneuver by taking a deep breath:The nerves Asambthawayh and nerves Albarasmbthawayh is regulating heart rate and strength of contractions (known as nerve Ha'ir) when the heart beats, the network will be Asambthawayh is dominant (a device that requests from the body mainly to accelerate), and what you should do is to shift control to the network nerves Albarasmbthawayh quieter, if affected vagal you begin the process of chemical rush of heart in the same way that trample on the brake the car to affect the speed.Another way to do this is to take a deep breath.Try to reach the carotid artery through rubbed:It is true that the right carotid artery massage is another way to vagal maneuver. Make sure that your doctor may show you the correct position and the right degree of pressure.You must be massaged at the confluence artery in the neck, under the jaw as far away as possible.Recall the reaction of the body to dive by immersing the face in ice water:When delve marine mammals in the coldest areas of water, Itbatye, heart rate automatically, and this natural way to keep the brain and heart animal, and can summon a reaction your body for diving in cold water so that fills a basin with iced water and immerse your face in it for a second or two.Stopped taking coffee, cola, tea and chocolate, slimming pills or stimulants in any form:The excessive intake of stimulants can expose you to the risk of fast heartbeat paroxysmal atrial fibrillation.Be sure to take care of the hypothalamus through proper diet and exercise:What is happening inside your head - specifically the hypothalamus - governs the heart - and this is why it is important to give the necessary attention to this part of the brain, through food الناسب and exercises to maintain stability and control of the future of the nervous system.The nervous system of the future is divided into two subgroups: the sympathetic nervous system future, which speeds up everything in the body except digestion, and the nervous system Albarasmbthawy.The tension and malnutrition and pollutants can make the hypothalamus loses control of the autonomic nervous system and allows the device to speed up the so-called sympathetic overload device future.You can help the hypothalamus in maintaining control.Eating a healthy diet and regular and reduced the dessert:If neglected some meals and then filled the stomach with candy or soft drinks, the pancreatic enzymes will increase to interest the amount of sugar large addressed, then increases insulin than normal and become a lack of sugar, and start glands adrenaline secretion of adrenaline to mobilize glycogen stores in the liver and then cause adrenaline a sudden increase in heart rate and a feeling of panic.You have to adapt the meals according to the table metabolismIt should people who have fast metabolism can eat foods rich in protein. The protein foods take longer to be digested and help prevent low blood sugar, when low blood sugar, it causes a process that we have discussed previously.You calmObserved a relationship between individuals who are characterized request perfection and who want height and repeat success and rapid heartbeat paroxysmal atrial fibrillation. Generally, these are people who have migraines, and for such, the mechanisms heart become exaggerated a lot, no overeating of chronic secretion of adrenaline, when exposed people to pressure nervous tremendous progress is made a surprise palpitations rapid and disappears irregular rhythm.How is compensation?You to follow a progressive relaxation program and dynamic exercise relax or learn how to imagine the serenity and calm, tranquility and security.Just eat yours from magnesiumThe magnesium protects cells, and for heart muscle cells, magnesium helps to offset the effect of calcium. When entering calcium into cells, it stimulates contractions of muscle within the same cell, and is magnesium necessary for enzymes that pumps calcium out of the cell, it causes constriction and relaxation in regular rhythm, it also makes the heart less susceptible to turbulence, and magnesium found in foods such as soy, nuts and peas, bran.Be careful on the high level of potassiumThe potassium is a mineral that helps to slow down the heart rate and uncontrollable muscle fibers, and no metal in some fruits and vegetables, and therefore obtain a sufficient proportion of it is not difficult. But you can Tstnfzh if your diet is rich in sodium, or if you are taking diuretics or use excessive laxatives.March exerciseYou can improve a lot of exercise, when doing exercises that increase heart rate, then return the heart to slow down the rate of pulses.The people who do not exercise have heart rate have about 80, and when they begin to practice some light exercises increase heart rate even up to 160, 170, and then after a short period of exercise, can be downloaded rate pulses to 60 and 65.The exercise will make you resist starting adrenaline overload.Like you graduated properly Tanatk, because you are using a starting adrenaline as part of their normal work.

Physical properties of blood

Physical properties of blood

Definition of blood Blood DefinitionBlood is a liquid texture, forms of connective tissue is happening inside the body within blood vessels (veins and arteries and capillaries) and consists of a liquid (plasma Plasma) swim blood cells Blood Corpuscles.Physical properties of bloodColor:Red color of blood and the presence of hemoglobin which gives blood on this color, red color varies in arteries than in veins is bright red in the arteries due to the presence of oxygen and dark red in the veins due to the presence of carbon dioxide.Temperature:It is fixed in the body, with some differences which from one member to another as needed for this member of the heat in order to do its main function. For example, the temperature of the liver equal (40-41) degree, either brain The degree temperature equal to less than 36 degrees Celsius.The overall rate of body temperature ranges between 36.8-37.8 degrees Celsius.Blood density:It relies on the presence of substances dissolved in the plasma, such as red blood cells, protein and value for men ranging from (1.057-1.067) g / cm 3.And women ranging between (1.051-1.061) g / cm 3.Blood viscosity:It is a friction force blood walls of the arteries and veins which mainly depend on the proteins found in plasma, especially Alvebrnugen and is important in maintaining blood pressure, a men's 4.7 and women 4.3.Osmosis:And this pressure results from the presence of crystals and minerals in the plasma, due importance to maintain the equivalent of salts and water inside and outside the cell (in the arteries and microvascular).For example, the presence of salts in abundance in the blood causes the withdrawal of water from the cells and this leads to dehydration, but the lack of salt water that causes entry into cells and this is known as water poisoning.
Pressure Alkoloido Asmosze (colloidal):And this pressure results from the presence of proteins in the plasma which is equal to 25 mm Hg and its importance due to:A) to maintain the presence of water within the vascular (blood volume).B) the exchange of nutrients between the blood and cells.A lack of protein in the blood leads to a lack of pressure Alkoloido and Asmosze which keeps water inside the cell and spoke edema Oedema and this pressure is less than the osmotic pressure normally.Density hydrogen concentration in the blood (PH):Usually this density tend to basal (ie blood basic solution) and is equal to this intensity of 7.4 and 7.35 in the arteries veins.The inside the cell is equal to (7-7.2) due to the presence of carbon dioxide.

Blood components

Blood components

 
1 - plasma plasmaIt is the liquid portion of blood, swim where blood cells and blood plasma ratio to total blood volume of 54%.Plasma properties:1. Color:Tends to yellow color and because of the presence of bilirubin Bilirubin.2. Figure:Plasma shapeless.3. Density:Plasma density is 1.027 g / cm 3, and it depends on the plasma proteins.4. The rest of the properties:(Viscosity / osmotic pressure, PH) as mentioned in the properties of blood.Blood plasma components:Blood plasma is composed of the following elements:• Water and accounts for 90% of the plasma volume.• solids and constitute 10% of the plasma volume, including:- 9% organic material.- 1% inorganic materials.1. Organic materials Organic materials:And this in turn is made up of:* Protein (6-8)% of the plasma volume (6-8 g / 100 cm 3 plasma).They are divided into:A - albumin Albumin and constitute 55% of the plasma proteins and this is equal to (8.3 to 1.5) g / 100 cm 3 plasma.B - globulin Globulin and accounting for 38% of the protein and this is equal to 3 g / 100 cm 3 plasma.C - Fabrinogen Fibrinogen and constitutes 7% of the protein and this equals (200-400) mg / plasma 100 cm 3.* Non-protein materials and are divided into:Food items, namely:- Sugars Glucose and increase of 80-120 mg / plasma 100 cm 3.- Lipid fats and increase of 600-800 mg / plasma 100 cm 3.Akhrajah materials such as:- Urea and urine increase of 11-53 mg / plasma 100 cm 3.- كرياتنين Creatinine increase of 0.8-1.2 mg / plasma 100 cm 3.- Uric acid Uric acid increase of 0.3-0.7 mg / plasma 100 cm 3.2. Inorganic materials Non organic materials:It includes:- Potassium and potassium 5.3 to 5.5 mg per Aikhuevilant / liter MEq / L- Sodium and sodium 135-153 mg per Aikhuevilant / liter.- Calcium and 8.8 -10.2 mg / plasma 100 cm 3.- Manganese and magnesium per 6.1 to 5.2 mg / plasma 100 cm 3.- Iron and iron rate of 100-150 mg / plasma 100 cm 3.- Chlorine and 38-110 mg per Aikhuevilant / liter.- Albaakrbunat.
2 - Blood cells Blood CellsAnd these cells are classified into:
Blood cells called because it does not contain recipes of cells where they contain a nucleus and nucleolus, Raibusomat and mitochondria so they are unable to divide and multiply.Numbering (5.4 to 5.6) million globule / mm 3 blood.
Blood cells in every sense of the word and number (4-11) A cell / mm 3 blood.• platelets Blood platelets.Circular objects do not carry recipes normal cell numbering (150-400) A plate / mm 3 blood.

Blood Circulation

Blood Circulation
Occur Arab world, Ibn al-Nafis (1210-1280) for the first time for micro-circulation and intended to promote blood circulation between the heart and lungs.After nearly four centuries came the British in 1678, "William Harvera" to talk in detail about great session and meant the circulation of blood between the heart and the rest of the body.The blood on a whirlwind tour of the human body, to reach every cell of it, carries the oxygen and food of any life, and carries waste, produced by and get rid of them through their work. On top of this waste carbon dioxide, which in turn get rid of it blood through a process of purification and cleaning take place in the lungs.And the blood passes through tours in the blood vessels of different capacities, size and composition.Called the vessels that carry blood from the heart to the other members of the body of the arteries, while called vessels that carry blood from the body to the heart veinsEveryone shall be two sessions: the bloody cycle or the circulatory system and other lymph.The circulatory system is divided blood or circulatory system into three parts all working at the same time:1 - Course pulmonary or Minor2 - plenary session or major session3 - Casement sessionMicro Valdorh or pulmonary circulation mobility blue blood to the lungs where it gets rid of carbon dioxide and provide themselves with oxygen. This is because blood red in color and loaded heart Booxgen.The plenary session of pure blood transfusion Red to the tissues.In textile filaments leaves blood carries oxygen and receives carbon dioxide and waste, to take her to and from the heart to the lungs through micro-session, which replaces the oxygen-carbon dioxide.The session Casement (PORTAL-CIRCULATION) are blood circulation to the liver, where moves blood received from the spleen, pancreas, stomach and intestines, and his liver converts glucose in the blood to glycogen and kept in reserve, come out of it in the case of need the body to him, as he turns Residue for cellular metabolism Albrootidi acid, urea, so The kidneys Bafrazha with urine.The center occupies the heart's main blood circulation. Reference is first and last a pump that receives blood and you pay to a permanent movement of vessels not only end with the end of life.Off blood from the heart, specifically the right part of it any of the right ventricle, through the pulmonary artery and pulmonary artery carrying blood saturated with carbon dioxide to the lungs Vivra to two major left and right.And branched These, in turn, to the branches reach each and every one of them to a certain lobe of the lung.And continues to branch like the branches of a tree even turned into a large network in harmony with lung Onsak (lung vesicles) Alveoles, where it is the process of purification and gas exchange.Gives blood how it surplus carbon dioxide and takes the oxygen it needs and returns through the veins, which are formed in a manner adverse to branch arteries, even to take final shape and make up four pulmonary veins carry blood saturated with oxygen to the heart, specifically the left atrium.This process is called the whole circulatory micro or small.What is different small for large session?Is essentially different from the major arteries that carry blood blue blood pregnant to carbon dioxide while carrying red blood veins purified oxygen dreamer, but in the session the major arteries that carry blood red while carrying blue blood veins.Off major blood during the session from the left ventricle through the aorta to the various parts of the body where branching artery bypass multiple names vary according to members that link to it and continue Baltafra even small branches up to every cell in the body.Up blood if, to the members of the body are all loaded with oxygen and return carrying carbon dioxide and other waste to accumulate through the veins that are formed in the same way that we have seen in the session Minor, and carry it to the heart through and Reden key are called veins Alogeoffan veines caves, one inferior vena cava vena cava inferior , gather the blood from the lower limbs to take it to the right atrium in the heart, while blood accumulates from the upper limbs by superior vena cava vena cava superior in turn also hurt in the right heart atrium.From there off to the right ventricle, where pumped through the pulmonary artery to the lungs to begin Minor new session.


It should be noted that the blood is subject during its grand to the purification process is in the kidneys, where blood is free from some of the material excreted with urine through a complex process of absorption and excretion.As is also subject to the process of purifying the other is in the liver through the cycle generic form part of the session the major, and are made through the portal vein (portal vein), which is transmitted through which blood coming from the intestines and abdominal organs other and which also carries food gained from foods that are undergoing liver where it is receive. فالكبد as we know is the chemical lab in the body.The sessions Aldmoatin Mvsoltan completely from each other only in some diseases that remain a hole or more between the wall of the atrium or the right ventricle Tsalhma Bashbahiama Aloasrin.Some of these diseases lead to congenital cyanosis, and sometimes remain the link between the pulmonary artery and the artery Alarbehr, called the ductus arteriosus, impassable which also leads to mixing of blood blue blood red.

Acute Lymphocytic Leukemia

Acute Lymphocytic Leukemia


Illustration of the immune system


Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, however, the bone marrow produces abnormal white blood cells. These cells crowd out the healthy blood cells, making it hard for blood to do its work. In acute lymphocytic leukemia (ALL), there are too many of specific types of white blood cells called lymphocytes or lymphoblasts. ALL is the most common type of cancer in children.
Possible risk factors for ALL include being male, being white, previous chemotherapy treatment, exposure to radiation, and for adults, being older than 70.
Symptoms of ALL include:
  • Weakness or feeling tired
  • Fever
  • Easy bruising or bleeding
  • Bleeding under the skin
  • Shortness of breath
  • Weight loss or loss of appetite
  • Pain in the bones or stomach
  • Pain or a feeling of fullness below the ribs
  • Painless lumps in the neck, underarm, stomach, or groin
Tests that examine the blood and bone marrow diagnose ALL. Treatments include chemotherapy, radiation therapy, stem cell transplants, and targeted immune therapy. Once the leukemia is in remission, you need additional treatment to make sure that it does not come back.
NIH: National Cancer Institute

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Book on Biological Laboratory penalty 17

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Policy & Procedure Manual
Policy # MI\TECH\48\v01  Page 3 of 3
Technical Manual   

Precautions

1.  The TSI tube should be read within 18-24 hr.  If read earlier, a false +/+ reaction may occur;
if after 24 hr, a fals e -/-reaction may occur.

2.   An H2S organism may produce so much black precipitate that the acidity in the butt is
completely masked.  If H
2
S is produced, an acid cond ition exists in the butt.

3.  There is no inhibitor in this  medium, therefore any organism  may grow.  Be certain that the
organism tested is a  catalase positive, gram  negative bacillus.

4.  S. typhi  usually produces a ring of H
2
S near the surface of the butt.  Occasionally the amount
of H
2
S produced is so small that it  will not be detect ed in TSI, but will show up in SIM
media.

5.  Some organisms produce such an abundance of gas that th e medium may be completely
displaced by gas, resulting in the medium being blown up into the cap of the tube.  Use
caution to avoid contamination.

6.  Do not tighten the cap of a TSI tube.  A free exchange of air is necessary to enhance the
alkaline reaction of the slant.

Quality Control

Test the media each  time it is prepar ed using the following organisms:

E. coli:    (ATCC 25922) :  +/+
  P. mirabilis:  (ATCC 12453) :  -/+/H2
S
  P. aeruginosa: (ATCC 27853) :  -/-

References

1. MacFaddin JF, Bioche mical Tests for Identifi cation of Medical Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p183-194.

PROCEDURE MANUAL
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Policy #MI\TECH\49\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Tube Coagulase Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

TUBE COAGULASE TEST
Principle

This test is used to speciate staphylococci by determ ining the ability of an isol ate to clot plasma by
producing the enzyme coagulase.

Reagents

Rabbit plasma 

  1.  Reconstitute one vial at  a time with sterile distilled water (volume determined by
vial size).

  2.  Store refrigerated before  and after reconstitution.   Use within 72 hours of
reconstitution.

Other Materials

Sterile glass tubes (tube method)
Culture loop or wooden applicator stick

Procedure

  1.  Add 0.5 mL of  plasma to a sterile glass tube.

  2.  Emulsify a large loop ful of a pure colony of  Staphylococcus into the plasma.

  3.  Incubate at 35
o
C for 4 hr, observing every 30  minutes for clot formation.

4.   If there is no visible clot at the end of 4  hours, leave at room temperature overnight
and observe for cl ot formation.

Interpretation

  Positive: Clot formation

  Negative: No clot formation

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Precautions

1)  When observing the tube, do not shake or agitate the tube.

Quality Control

Each time a coagulase test is performed, known  positive and negative cultures must be tested.

 Positive: S. aureus  (ATCC 25923)
 Negative: S. epidermidis  (ATCC 12228)

References

1.  MacFaddin, J.F., Biochemical Tests for Identification of Medical Bacteria, 2nd ed.,
Williams and Wilkins, Baltimore MD, 1980, pgs. 64-77.

PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\50\v01  Page1 of  2 
Section: Technical Manual  Subject Title: Urea Slant
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

UREA SLANT

Principle

To determine the ability of an organism to split urea by the action of the enzyme urease forming
two molecules of ammonia with resulting alkalinity.

Materials

Urea Slant
Bacteriology loop

Procedure

1.   From one isolated colony, heavily inoculate the urea slant.
2.   Incubate O
2, 35
0
C.
3.   Read at 3 hours and ag ain at 18-24 hours.

Interpretation

Positive: Intense pink-red  colour.
    Rapidly positive:  1 to 6 hours (Proteus spp.)
  Delayed positive: ≥ 18 hours

Negative: No colour change

Precautions

Urea test media rely on the demonstration of alkalinity, thus are not specific for urease.  The
utilization of peptones or other prot eins may cause an increase in pH.

Quality Control

Controls should be set up weekly.

P. mirabilis  (ATCC 12453):    Positive - 4 hours
  K. pneumoniae (ATCC 13883):  Weak positive - 18 hours
  E. coli  (ATCC 25922):    Negative

PROCEDURE MANUAL
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Technical Manual   

References

1.  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2
nd
 ed.,
Williams and Wilkins, Baltimore MD, 1980, p298-308.


PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\51\v01  Page 1 of  2
Section: Technical Manual  Subject Title: X And V Disks for
  Identification of Haemophilus
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

X AND V DISKS FOR IDENTIFICATION OF HAEMOPHILUS

Principle

Haemophilus spp. have different requir ements for the growth factor s X (hemin) and V (NAD).
These requirements are determined based on th e presence or absence of growth around disks
impregnated with V,X and XV factors.

Reagents

1.  Bacto Differentiation Disks
    BV  NAD and 125 units/ml bacitracin
    BX  hemin and 125 units/ml bacitracin
    BVX NAD, hemin  125 units/ml bacitracin
  Store refrigerated

2.  Mueller Hinton Agar (MHA)

Other Materials

Forceps
Swabs
Inoculating loop

Procedure

1.  Pick one colony from CHOC, taking care not to carry over  any agar from the medium.
2.  Inoculate MHA and streak  over the entire surface of th e plate using a sterile swab.
3.  Place X, V and XV disks on the surface of the agar in the form of a triangle with at least 30-35 mm between disks.
4. Incubate CO
2
, 35
o
C X 18-24 hr.
5.  Examine the pattern of growth around and/or between the disks.

Interpretation

Growth around the V and XV or the X and XV indicates a requirement for the single growth factor
V or X respectively.  Growth around only the XV disk indicates a requiremen t for both factors.

PROCEDURE MANUAL
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Precautions

1.  Avoid carry-over of growth factors.

Quality Control

Known positive and negative controls must be set up in parallel with the test.

H. influenzae (ATCC 35056):    Growth  around the XV disk only
H. parainfluenzae (ATCC 7901):   Growth around V and XV disks

References

1. Difco Package in sert, June 1984.


PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\52\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Xylose Fermentation
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

XYLOSE FERMENTATION
Principle

A rapid chromogenic test for the identification of  E. gallinarum.

Reagents

Prolab d-xylose tablets.
Sterile staline.

Other Materials

Sterile tubes (13 x 100 mm)

Procedures

1.   Suspend the growth from BA in 0.25 mL salin e to achieve the turbidity >#2 McFarland
standard.
2.   Add 1 tablet to the tube.
3.   Incubate at 35 - 37
0
C x 2 hours.
4.   Examine the tube for development of a yellow colour.

Interpretation

Positive:  Yellow / yellow orange colour
Negative: Red

Quality Control

The following organisms are tested weekly:

 Positive: E. gallinarum (ATCC 35038)
 Negative: E. faecalis (ATCC 29212)

References

1.  J. Clin. Microbiol. 12, 620-623, 1980.

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Book on Biological Laboratory penalty 16

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\46\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Thermonuclease Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

THERMONUCLEASE TEST
Principle

Staphylococcus aureus  contains a heat-stable thermonu clease and coagulase negative
staphylococcus does not.  This is a rapid test to differentiate between the two organisms.

Materials

Toluidine blue-O  DNA plate (Q-Lab)
13x100 mm tube with white cap
pasteur pipettes

Procedure

1.  Dispense 2 - 3 mL of blood  broth from BacT/Alert bottle  showing gram po sitive cocci in
clusters in the direct Gram stain into a sterile capped 13x100 mm tube.
2.  Place tube in heating block, 100
0
C for 15 minutes.
3.  Let cool to room temperature.
4.  Centrifuge at approximat ely 2500 rpm for 3 minutes.
5.  Inoculate a pre-warmed (35
o
C for 1 hour) toluidine blue-O  DNA plate by filling wells (cut
well with the end of a pasteur pipette) with 2 drops of the supernatant.
6.  Incubate the plate at 35
o
C in the upright positio n (agar side down).
7.  Inspect the plate at, 1 hour, 2 hours and 4 hours and again  after overnight incubation if
negative at 4 hours.
8.  Always run negative and positive cont rol wells with each  plate each day.

Interpretation

Positive:  Pink zone of clearing at the edge of  the well with a darker  blue ring at the outer
periphery of the zone;  indi cates thermonuclease activity

Negative:  No zone or a small clear zone around the well

PROCEDURE MANUAL
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Technical Manual   

Quality Control

1.  Inoculate 5 day negative patient BacT/Alert bottles with 0.5 mL of  a slightly turbid
suspension of (a)  S. aureus  (ATCC 25923) and (b)  S. epidermidis  (ATCC 12228) in
trypticase soy broth.

2. Incubate the bo ttles overnight at 36
o
C on the shaker.

3.  Remove 3 - 6 mL of the broth-blood from the bottles and process in the same manner as the
patient specimens (steps 1 to 4). Always QC new controls before use with patient specimen.

4.         Supernatants may be kept refriger ated for up to 1 month for use as controls.

Reference

1.  Rafner, H.B., & Stretton C.W.  1985.  Thermonuclease test fo r same day identification of  S.
aureus  in blood cultures.  J.  Clin. Microbiol. 21:995-996.


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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\47\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Tributyrin Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

TRIBUTYRIN TEST
Principle

A rapid chromogenic test for the identification of M. catarrhalis.

Reagents

Prolab Tributyrin (TRIB) tablets.
  Store at room temperature.
Sterile saline.

Other Materials

Sterile tubes (13 x 100 mm)

Procedures

1.  Suspend the growth from C HOC in 0.25 mL (6 drops) saline to achieve the  turbidity >#2
McFarland standard.
2.  Add 1 tablet to the tube.
3.  Incubate at 35
0
C x 4 hours.
4.  Examine the tube for development of a yellow colour.

Interpretation

Positive: Yellow/yellow orange colour

Negative: Red

Quality Control

Test the following organism weekly:

Positive:  M. catarrhalis  (ATCC 8176) 
 Negative: N. gonorrhoeae (ATCC 43069)
 
References

1.  Prolab package insert, February 1985.

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\48\v01  Page 1 of  3
Section: Technical Manual  Subject Title: TSI (Triple Sugar Iron)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

TSI (TRIPLE SUGAR IRON)
Principle

To determine the ability of an organism to attack  a specific carbohydrate  incorporated in a basal
growth medium, with or without  the production of gas, along with  the determination of possible
hydrogen sulfide (H
2
S) production.  This test is used,  in conjunction with others, for the
identification of enteric pathogens.

Materials

TSI Slant
Inoculating wire or sterile glass pasteur pipette.

Procedure

1.  Using the an inoculating wire, dip into the previously inoculated TSB.
2.  Stab the butt of the TSI to within 1/4 inch from bo ttom, draw out and fishtail over slant.  Do
not tighten cap.
3. Incubate O
2, 35
o
C X 18-24 hours.

Interpretation

Carbohydrate utilization:
  1.  Fermentation of glucose only
    (a)  slant:  red colour (alkaline reaction)
    (b)   butt:   yellow colour (acid reaction)

  2.  Fermentation of glucose and sucrose and/or lactose
    (a)  slant:  yellow colour (acid reaction)
    (b)  butt:   yellow colour (acid reaction)

  3.  Neither glucose nor lactose nor sucrose fermented
    (a)  slant:  red colour (alkaline reaction)
  (b) butt: (i)  aerobic organism
        (a)  No growth
        (b)  No colour change
 (ii) facultative organism       red colour (alkaline reaction)

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Gas production:

 1. Aerogenic:
  (a) Gas production:  CO2 and H
2

    (b)  Evident by one of the following:
    (i) a single gas bubble
    (ii) bubbles in the medium
             (iii)  splitting of medium
    (iv) complete displacement of the medium from bottom of the tube
leaving a clear area
        (v)  slight indentation of medium from the side of the tube

 2. Anaerogenic:
   No gas production
 
H2
S production:

      The presence of a black precipita te (ferrous sulfid e) is evident by:
(i)   A black colour spread throughout the entire butt masking the
            acidity;  may even  be a slight evidence  on the slant
     (ii) A black ring near the top of the butt area
  (iii)      A black precipitate scattered throughout  the butt  but not entirely
                        masking the acidity present
Summary:

  The ways of recording the TSI reactions are list ed below.  Reme mber that the slant is first,
followed by the butt reaction.

  acid/acid    +/+
  acid/acid/gas    +/+ with gas
  acid/acid/gas/H2S   +/+ with H 2
S
  alkaline/acid    -/+
  alkaline/acid/gas   -/+ with gas
  alkaline/acid/gas/H2S      -/+ with gas and H2
S
  alkaline/acid/H2S        -/+ with H 2
S
  alkaline/alkaline   -/-


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5.   Apply coverslip to wetted slide and examine with the fluorescent microscope using the
designated filter.  If there is a delay, add distilled water to the coverslip just prior to
examination.  Use a fresh tube of water daily.

6.   Optional for thicker smears .  Add few drops of the counter stain Fungi-Fluor solution B.
Rinse gently with tap water and th en proceed as in step 5 above.

Quality Control

Stain a smear of  Candida albicans daily.

Interpretation

Use 20x or 40x objective.

Fungal elements will appear yellow-green agains t a red-orange background  when counterstain is
used.  Observe for characteristic morphology.

References

1.   Manufacturers' Instructions (Data Sheet #316).  Fungi-Fluor  kit - Polysciences, Inc.,
July 1995

2.   Clin. Micro. Newsletter 9:33-36, March 1, 1987.
K.L. McGowna.  "Practical Approaches  to Diagnosing Fungal Infections in
Immunocompromised Patients".

3.   J. Clin. Micro. 28:393-394, Feb. 1990.  V.S. Baselski et al.  "Rapid Detection of
Pneumocystis carinii in Bronchoalveolar Lavage Sample s by Using Cellofluor Staining".


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Section: Technical Manual  Subject Title: Gram Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

GRAM STAIN

Principle

Bacteria can be recognized as gram positive (blue-black/purple) if they retain the primary dye
complex of crystal violet and iodine in the face of attempted decolourization, or as gram negative
(pink) if decolourization occurs as shown by the cell accepting the counterstain safranin.

Generally the mechanism of the Gram stain is:  The fixed bacteria are stained with the
triphenylmethane dye, crystal violet.  Next the smear is flooded with Grams solution which
oxidatively forms an insoluble complex with the crystal violet.  The smear is then flooded with
the organic solvent, acetone-alcohol.  Dependi ng on cell permeability the  crystal violet-iodine
complex will be washed from Gram negative bact eria in solvent but not from Gram positive
bacteria.  Upon counterstaining with safranin,  organisms which had been  discolorized by the
ethanol (Gram negative) will stain pink.  Gram positive organisms which retained the crystal
violet will appear blue-black/purple microscopically.

Materials

Crystal violet solution
Grams Iodine solution
Acetone alcohol
Safranin solution

Procedure

1.   Prepare the film on the slide and allow to air dry.
DO NOT HEAT TO DRY FILM.

2.   When film is dry, place slide on heating bloc k for several minutes.  Slide should be just
warm to your hand.
DO NOT OVERHEAT.

3.   Allow slide to cool - this will happen quickly  - in just a few seconds.
DO NOT ADD STAIN TO HOT SLIDE.

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4.   Flood slide with crystal violet - leave 1 minute.

5.   Wash gently with water.

6.   Flood slide with Grams Iodine - leave 1 minute.

7.   Wash iodine from slide with acetone-alcohol mixture.  Add a few more drops of acetone-alcohol until no more colour comes from film - usually 30 seconds.

8.   Wash gently with water.

9.   Flood slide with safranin - leave 1 minute.

10.  Wash gently with water.  Clean back of slide with tissue and  place slide in tray.  

Precaution

1.   At no time should the film (smear) be exposed to too much heat.  When the specimen is
still wet, heat causes coagulation of the  protein resulting in heavy overstaining which
cannot be removed by the decolourizer.  A thic k smear will also show more tendency to
"lift off" during staining.

2.   Rinsing the Grams Iodine off with the decolorizer gives more stability to the CV-GI
complex and false over decolorizing will not take place.

3.   Flooding a hot slide with crystal violet will cause the stain to  precipitate and make
decolourizing much more difficult.

Quality Control

It is recommended that controls  be run concurrently with unknowns or at least run on a daily
basis using known smears containing Gram positive and Gram negative bacteria.


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Section: Technical Manual  Subject Title: Staphaurex Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

STAPHAUREX TEST

Principle

A rapid slide latex test for the detection of clumping factor and prot ein A produced by most
strains of  S. aureus.

Reagents and Materials

Staphaureux latex suspensi on (store refrigerated)
Disposable reaction cards
Culture loop or wooden applicator stick

Procedure

1.  Confirm the identification of a suspect  Staphylococcus by Gram stain and catalase test.
2.  Allow the latex reagent to warm to room temperature before use.
3.  Shake the reagent so that all of the particles are resuspended.
4.  Dispense one drop of latex  reagent onto the reaction card.
5.  Add 1-3 colonies to the drop, mix well  with a loop or wooden applicator stick.
6.  Rock the slide for 20 seconds and look for clumping.
7.  Discard the slide into a discard container.

Interpretation

Positive test:  Clumping within 20 seconds  with the sensitized latex particles.
Negative test:  No clumping

Precautions

1.  False positive results may occur after 20 seconds.
2.  False positive agglutination can occur with   E. coli and 
  C.  albicans

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Quality Control

Test known positive and negative controls daily:

 Positive: S. aureus  (ATCC 25923)
 Negative: S. epidermidis  (ATCC 12228)

References

1.  Staphaurex Package insert, July 1992.


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Section: Technical Manual  Subject Title: Streptococcal Grouping
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

STREPTOCOCCAL GROUPING
Principle

This test is used to determine the Lancefield gro up of an isolate.  Latex particles labelled with
specific group antisera will agglutin ate in the presence of the corre sponding antigen after extraction
with nitrous acid.

Reagents

Pro-lab Streptococcal grouping Latex kit.

Other Materials

Droppers
Disposable slides
Wooden stirring sticks
13x100 mm test tubes

Procedure

1.  Label one test tube for each isolate.
2.  Add one drop of Extracti on Reagent 1 to each tube.
3.  Suspend 4 beta-haemolytic colonies in the Extraction Reagent 1.
4.  Add 1 drop of Extraction Reagent 2 to each tube.
5.  Shake the tube and inc ubate for 2 minutes at RT.
6.  Add 7 drops of Extraction Re agent 3 to each tube.  Mix th e reaction by vortexing the tube
for 10 - 15 seconds.
7.  Dispense one drop of each la tex suspension to be  tested onto separate  circles on the test
card.
8.  Using a pasteur pipette, add one drop of extract to th e latex suspension.
9.  Mix the latex and extract wi th the wooden stick using the co mplete area of the circle.
10.  Gently rock the card for 2 minutes and lo ok for agglutination.

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Interpretation

Positive: Strong vi sible agglutination  within 2 minutes.

Negative:  Milky appearance without visible agglutination.

Precautions

1.  False positive reactions have been known to occur with organisms from unrelated genera eg.
E. coli,  Klebsiella  sp.,  Pseudomonas sp.

Quality Control

Test reagents are checked weekly.

Each test should be tested with at least one extra grouping latex su spension as a negative control.

Reference

1. Pro-lab Streptococcal  Grouping package insert.

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Section: Technical Manual  Subject Title: Tetrazolium Reduction
 Test (TTC)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

TETRAZOLIUM REDUCTION TEST (TTC)

Principle

To differentiate between E. faecalis and  E. faecium.

E. faecalis reduces the colorless compound (Tetrazolium- chloride) to an insoluble substance -
formozan which is red.

Material

BHI broth/Tetrazolium-chloride (TTC)

Procedure

Inoculate a loopful of an overn ight plate culture to 1 ml of  TTC broth.  Incubate at 35
0
C and
observe the reaction at 2 hours.  If negative, reincubate  up to 8 hours / overnight.

Interpretation

Positive - deep magenta
Negative - colourless or faint pink

Quality Control

Positive:  E. faecalis   (ATCC 29212)
Negative:  E. faecium   (ATCC 19434)

References

1.   J. Gen. Microbiology (1965), 38, 279-287.
 

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Section: Technical Manual  Subject Title: Acid Fast Stain for
 Mycobacteria (Kinyoun)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN)

Principle

To stain Mycobacteria present in specimens  and cultures.

Mycobacteria are different to stai n with common aniline dyes.  However, they will stain with basic
fuschsin.  Once stained, they retain the dye de spite treatment wi th mineral acid s i.e.  HCl H
2SO4
.
This property of acid fastness  may be due to a lipd fraction called mycolic acid.  Mycobacteria also
exhibit degrees of resistance to  decolourization with alcohol.

Materials

Kinyoun Carbol fuchsin
3% HCl in 95% ethanol
Brilliant green

Procedure

1.   Prepare smear over an  area of 2-3 sq. cm.

2.   Heat fix smear on heating block (56
0
C/1 hr).  Then hold to  incinerator for 10 secs.

3.   Place slide on stain rack and allow to cool.  Flood with Kinyoun Carbol fuschsin for 5 min.

4.   Rinse off stain with water.

5.   Decolourize with 3% acid alcohol for 3 mins.

6.   Rinse with water.

7.   Repeat decolourization for 1-2  mins. or until no red appears.

8.   Rinse with water.

9.   Flood slide 3-4 mins. with Brilliant green.

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10.  Rinse with water.

11.  Air dry.  DO NOT BLOT.

Microscopy

Place a drop of oil between the specimen and coverslip and oil again on top.  Smears are examined
with oil immersion lens.  The coverslip prevents cr oss contamination.  An average of 15 mins. is
spent on each slide.  The total area of the spec imen must be examined.

References

1.   Baker, F.J., Breach, M.R.   1980.  Medical Microb iological Technique, p. 15


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Section: Technical Manual  Subject Title: Acid fast stain for Nocardia
(Modifed Kinyoun)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN)

Principle

Nocardia  species possess the unique characteristic of resisting d ecolorization with acid alcohol.

Reagents

1.   Carbol-fuchsin
Basic fuchsin solution   (3 g basic fu chsin in 100 mL 95% ethyl alcohol)
10 mL
Phenol 5% aqueous    90 mL

2.   Decolourizer (1% sulfuric acid)
H2 SO4
 (concentrated)   1 mL
Distilled water   99 mL

3.   Methylene blue
Methylene blue    0.3 g
Distilled water   100 mL

Staining Procedure

1.   Fix the smear by gentle heating.
2.   Flood the smear with Carbol fuchsin solution.
3.   Allow the slide to stand for 5 minutes.
4.   Wash the smear with tap water.
5.   Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing
(approx. 1 min.).
6.   Rinse with tap water.
7.   Counterstain with methylene blue about 1 minute.
8.   Rinse with tap water and air dry.

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Interpretation

The filaments of  Nocardia  species and Rhodococcus appear red-stained against a blue
background.

Quality Control

A positive control slide of Nocardia species is stained simultaneously with the clinical
specimens.

References

1.  Murray, PA. et al.  Manual of Clinical Microbiology, 7
th
 edition, 1999 ASM,
Washington, D.C.

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Section: Technical Manual  Subject Title: Acridine Orange Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACRIDINE ORANGE STAIN
Principle

Acridine orange is a fluorescent dye which will bi nd to the nucleic acid of  bacteria and other cells.
It is recommended for use  for the detection of microorganisms in direct smears.  It is useful for the
rapid screening of specimens from  normally sterile site s (eg. CSF) and blood smears, or smears
containing proteinaceous material where differentiation of organisms from background material
may be difficult.   

Reagents

Acridine Orange spot test dropper.  Stored at room temperature.
Absolute Methanol

Procedure

1.  Prepare a smear of the  specimen to be stained.

2.  Allow to air dry.

3.  Fix with methanol for 1 to 2 minutes.

4.  Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.

5.  Flood the slide with the acridi ne orange and stain for 2 minutes.

6.  Rinse thoroughly with tap water and allow to air dry.

7.  Examine with a fluorescent microscope using low and oil immersion objectives.

Interpretation

Bacteria and fungus stain bright orange.  The background appears black to yellow green.
Leukocytes will stain yellow, orange and red.

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Quality Control

Stain a smear of Streptococcus pneumoniae (ATCC 6303)  each time the  test is performed.   

References

1.  Difco Spot Test Acridine Orange Stain package insert, 1984.

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Section: Technical Manual  Subject Title: Bacto 3-Step Gram Stain
 Procedure
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

BACTO 3-STEP GRAM STAIN PROCEDURE

Principle

To be used for problem smears to determine the Gram reaction of organisms.

Materials

3-Step Stabilized Iodine Technique

Bacto Gram Crystal Violet
Bacto Stabilized Gram Iodine
Bacto 3-Step Gram Safranin-S

3-Step Technical Iodine Technique

Bacto Gram Crystal Violet
Bacto Gram Iodine
Bacto 3-Step Gram Safranin-T

Microscope slides
Bunsen burner or methanol
Bacteriological loop
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol  Gram Slide
Bactrol  Disks

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Procedure

1.   Flood the fixed smear with primary stain (B acto Gram Crystal Violet) and stain for 1
minute.
2.   Remove the primary stain by gently washing with cold tap water.
3.   Flood the slide with mordant (Bacto Stabilized Gram Iodine or Bacto Gram Iodine
(traditional formulation) and retain on the  slide for 1 minute.  (Refer to LIMITATIONS
OF THE PROCEDURE, #5)
4.   Wash off the mordant with decolourizer / counterstain (Bacto 3-Step Gram Safranin-S or
Bacto 3-Step Gram Safranin-T).  ( NOTE : Do not wash off iodine with water).  Add more
decolourizer / counterstain solution to the slide and stain 20-50 seconds.
5.   Remove the decolourizer / counterstain solution by gently washing the slide with cold tap
water.
6.   Blot with blotting paper or pa per towel or allow to air dry.
7.   Examine the smear under an oil immersion lens.

Interpretation

REACTION   3-STEP TECHNIQUE
using either Bacto Gram Safranin-S or Bacto
Gram Safranin-T

                       Gram-positive

                       Gram-negative
   Purple-black
to purple cells

Red-pink to
fuchsia cells

Quality Control

Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative
microorganisms. 

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Section: Technical Manual  Subject Title: Eosinophil Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

EOSINOPHIL STAIN
Principle

A stain for the detection of eosinophils in clinical specimens.

Reagents

AJP Scientific Eosinophil stain:
  Solution I - Eosin Y
  Solution II - Buffer Ph 6.5
  Solution III - Methylene Blue
  Stored at room temperature

Procedure

1.  Make a thin smear and spread evenly.

2.  Fix slide by air drying  or with gentle heat.

3.  Cover slide with solution I and leave for 30 seconds.

4.  Add solution II to cover s lide.  Mix gently  and allow to stain for 3 to 5 minutes.

5. Wash off with  tap water and drain.

6.  Cover slide with solution III and immediately  wash off with tap wate r.  Drain and air dry.

Interpretation

Eosinophils stain with red cytoplasm and bright red granules.

Reference

1. A.J.P. Scientific INC package insert.

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Section: Technical Manual
Subject Title:  Fungi-fluor   Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

FUNGI-FLUOR STAIN

Principle

The Fungi-Fluor  stain is used for the rapid identification of various fungal infections in fresh or
frozen clinical specimens.

The active, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of
4,4'-bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-tr iazin-2-ylamino]-2,2'-stilbenedisulfonic acid.
Fungi-Fluor  staining solution is a 0.05% solution of this  dye in deionized water with potassium
hydroxide added as a clearing agent.  The Fungi-Fluor  counter staining solution B is an
aqueous solution of Evans Blue dye used to re duce background fluorescence.  Cellufluor binds
nonspecifically to beta-linked polysaccharides found in the cell walls of various organisms such
as chitin and cellulose.

Materials

Staining Solution A
Counterstaining Solution B
Absolute alcohol
Water
Fluorescent Microscope (250-400 nm filter)

Precautions

1.  Store in a dark or opaque bottle, tightly sealed, at room temperature.

2.  Avoid eye or skin contact: use gloves and protective glasses.

Procedure

1.   Prepare smear of specimen and allow to air dry.

2.   Fix on the rack with absolute alcohol for 5 mins. until dry.  Fixed smears can be held
indefinitely until ready to stain and examine.

3.   Add few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute.

4.   Rinse gently with tapwater.

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Section: Technical Manual  Subject Title: RapID ANA II System
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID ANA II SYSTEM

Principle

The RapID ANA II System is a qualitative micromethod employing conventional and
chromogenic substrate for the identification of  medically important anaerobic bacteria of human
origin.

The tests used in it are based upon the microbial degradation of specifi c substrate detected by
various indicator systems.  The reactions are a combination of conventional tests and single-substrate chromogenic tests.

Materials

1.   RapID ANA II panels
2.   Suspension fluid
3.   Kovacs spot indole reagent
4.   RapID ANA II reagent
5.   RapID ANA ID forms

Procedure

Make an equivalent McFarland #3 turbidity suspension of 18-24 hours AnO
2
 culture (not more
than 72 hours) in the supplied suspension fluid.  Mix it thoroughly - can be used up to 15
minutes.  Inoculate an agar (BA FAA) plate for purity and incubate for 24 hours anaerobically.
Peel the lid off the panel marked "peel to inoculate".  Using th e Pasteur pipette, transfer the
entire contents into the right uppe r corner of the panel. Seal the panel. Level the contents in the
panel and slowly tilt the panel so that every chamber receives  an equal amount of suspension.
Incubate the panel at least four hour s (not more than six hours) in non-CO
2
 incubator at 35-37
0
C.
After the incubation period, read the panel prior to adding the r eagents and write results on the
ID form.  Add the reagents as per instructions.  Allow 30 seconds  but not more than two minutes.
Read it and score on the form.

Interpretation and Identification

Please follow the guidelines from the manufact urer and see the RapID ANA II ID Code Book.

See RapID ANA II System Insert #iii08-1/94 brochure which follows.

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Policy & Procedure Manual
Policy #MI\TECH\38\v01  Page 1 of  2
Section: Technical Manual  Subject Title: RapID MGP Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID MGP TEST

Principle

Rapid MGP Medium is a 5 hour test for the differentiation of  Enterococcus faecium  and  E.
faecalis  from  Enterococcus gallinarum and  E.  casseliflavus based on the ability to acidify the
carbohydrate methyl-glucopyranoside (MGP).

Reagents

Rapid MGP Medium (Hardy Diagnostics)
Bacteriology loop

Procedure

1.   Using a sweep of colonies from an 18-24 hour  pure culture of the organism to be tested,
stab the MGP media with the loop.  There should be a visible cell paste on the loop as the
media is inoculated.

2.   Incubate aerobically at 35
0
C for 5 hours.

3.   Observe for the development of a yellow colour along the stab line  indicating a positive
test.

4.   Reincubate weak reactions for 24 hours.

Interpretation

Positive:  yellow colour along stab line
Negative:  colour remains blue

E. casseliflavus +
E. gallinarum   +
E. faecalis   -
E. faecium   -

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TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\38\v01  Page 2 of 2
Technical Manual   

Quality Control

Positive and negative controls are  run each time the test is set up.

Positive:  E. casseliflavus   (ATCC 12755)

Negative:  E. faecalis   (ATCC 19966) 

Reference

1.  Hardy Diagnostics package insert 1999.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\39\v01  Page 1 of  1
Section: Technical Manual  Subject Title: RapID VP Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID VP TEST

Purpose

To aid in the identification of  S. milleri.

Media

MR - VP broth

Procedure

1.   Transfer approximately 0.2 ml of VP broth into a sterile 13 x 100 test tube.

2.   Using a sterile inoculating wi re inoculate the test organi sm heavily into the broth.

3.   Incubate the tube at 35
0
C for 5 hours.

4.   After incubation add 1 drop of alpha-naphthol and 1 drop of 40% KOH.

5.   Shake the tube gently for one minute to expose the medium to air.  Allow 10-15 minutes
for reaction to develop.

Interpretation

 Positive - Red colour
  Negative  -  No colour change within 10-15 minutes

Precautions

The order of adding reagents is important; alpha-naphthol followed by 40% KOH.

Reference

Ruoff, K.L., Ferraro, M.J. 1986.  Presumptive identification of  S. milleri  in 5h J Clin Microbiol
24:495-497.


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TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\40\v01  Page 1 of  2
Section: Technical Manual  Subject Title: RapID Yeast Plus Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID YEAST PLUS TEST
Purpose

Used for the identification of  yeast and yeast like organisms.

Materials

Rapid Yeast Plus Panel
Rapid Yeast Plus Reagent A and Reagent B
Rapid ID Inoculating fluid 2ml
Pasteur Pipettes
Cotton swabs

Procedure

1.   Use a cotton swab suspend sufficient growth of the yeast in the Inoculating fluid to give a
suspension heavy enough to obliterate the black lines on the inoculation card.

2.   Peel back the panel lid over the inoculation port by pulling the tab marked "Peel to
inoculate".

3.   Using a Pasteur pipette transfer the entire contents  of the inoculation fluid into the upper
right hand corner of the panel and then reseal the panel.

4.   Tilt the panel back away from the biochemical wells at approx. a 45% angle.

5.   While tilting back gently rock the panel from side to side to evenly distribute the
inoculum along the rear baffles.

6.   Slowly tilt the panel forward toward the reaction cavities until th e inoculum flows along
the baffles into the biochemical wells.

7.   Incubate panel at 30
0
C for 4 hours.

8.   After incubation peel the label lid from over the reaction cavities.

9.   Add 1 drop of reagent A to cavities 7 to 14 inclusive.

10.  Add 1 drop of reagent B to cavities 16-18 inclusive.

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Policy & Procedure Manual
Policy # MI\TECH\40\v01  Page 2 of 2
Technical Manual   

11.  Read results after 30 seconds but no more th an 1 minute after addi ng reagent.  Record
results onto supplied report form, and look results up in the Rapid ID Yeast Plus code
book for interpretation.

Interpretation

Well # Positive Negative

1 to 5

Yellow  Blue to green
6

Yellow  Red, pink, orange, gold
7 to 14

Any yellow  Clear or cream
15

Red or dark red-orange  Yellow, yellow-orange or orange
16-18

Purple, red or dark pink  Clear straw, orange, pale to medium pink

Quality Control

Control strains are set up for each new lot number of panels.

References

1.  Rapid ID yeast plus package insert issue #7/98.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\41\v01  Page 1 of  2
Section: Technical Manual  Subject Title: SIM (Sulfide-Indole-Motility)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

SIM (SULFIDE-INDOLE-MOTILITY)
Principle

1.  To determine the ability of an organism to liberate  hydrogen sulfide (H
2
S) from sulphur-bearing amino acids pr oducing a visible, black colour reaction.
2.  To determine the ability of  an organism to split indole from the tryptophan molecule.
3.   To determine if the organism is motile or  non-motile.

This test is used, in conjunction with others, for the  identification of  Enterobacteriaceae  when
unable to identify usin g VITEK or API system.

Reagents

Kovac's Reagent

Other Materials

SIM Medium.
Inoculating wire or sterile glass pasteur pipette.

Procedure

1.  With a pasteur pipette, draw up a smal l amount of previously inoculated TSB.
2.  Stab vertically into the medium to within 1/4 to 1/2 inch fr om bottom: withdraw inoculating
needle following lin e of inoculation.
3. Incubate O
2, 35
o
C X 18-24 hours.
4.  Add a few drops of Kovac's reagent and  observe for development of a red colour.

Interpretation

H2
S production
  (a)  Positive:  any blackeni ng of the medium      
  (b)  Negative:  no blackening


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Policy & Procedure Manual
Policy # MI\TECH\41\v01  Page 2 of 2
Technical Manual   

Motility
 (a) Positive: motile organisms migrate from the stab line  and diffuse into the
medium causing  turbidity. They may exhibit fuzzy streaks of
growth.  (Compare with  an uninoculated tube.)
 (b) Negative: bacterial growth accentuated along stab  line; surrounding medium
remains clear.
Summary:

  Results are recorded as follows.  Remember that H
2
S is first, then  indole and finally
motility.
  -/-/- no H2
S, indole neg, non motile
  -/-/+ no H2
S, indole neg, motile
    -/+/-     no H
2
S, indole pos, non motile
    -/+/+     no H
2
S, indole pos, motile
  +/-/-    H2
S, indole neg, non motile
  +/-/+    H2
S, indole neg, motile
  +/+/-    H2
S, indole pos, non motile
  +/+/+   H2
S, indole pos, motile

  Refer to Manual of Clinical Microbio logy for specific organism reactions.

Precautions

1. An H
2
S-producing organism may exhibit blackening on SIM medium, but none on TSI
medium.
2. Some H
2
S inhibition occurs when  the temperature exceeds 34
o
C.
3.  Many bacteria are motile  at one temperature and non -motile when at another.
4.  If a motility test is difficult to interpret,  compare with an uninoculated motility tube.  If still
in doubt, perform a wet prep or hanging drop pr eparation using a heavy loopful of an 18-24
hr culture.

Quality Control

Quality control must be performed on each  new lot of SIM before bein g put into general use.
  K. pneumoniae (ATCC 13883):   -/-/-
  P. vulgaris (ATCC 13315):       +/+/+

References

1. MacFaddin JF, Bioche mical Tests for identifi cation of Medical Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p162-173, 173- 183, 214-218.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT