DE2129384A1 - Diagnostic preparation for the identification of hemolytic streptococci - Google Patents

Description

Dr. F. Zumstein Sr. - Dr. E. Assmann Dr. R. Koenlgsberger - Dlpl.-Phys. R. Holzbauer - Dr. F. Zumsteln Jun.

PATENT LAWYERS

TELEPHONE: COLLECTIVE NO. 225341 TELEGRAMS: ZUMPAT CHECK ACCOUNT: MUNICH 91139

BANK ACCOUNT: BANK H. HOUSES

8 MUNICH 2,

Case 54 277

WAHBER-LAMBEHT COMPANY, Morris Plains, New Jersey, USA

Diagnostic preparation for the identification of ß-hemolytic streptococci

The present invention relates to a preparation for the biochemical identification of ß-hemolytic streptococci in serological groups, ie in groups A, B, C, D. This preparation contains a carrier, ie a spongy strip that has a glycerin reagent band, a salicin reagent band and and Esculin reagent band contains. The glycerine and salicine reagent bands also contain pH indicator systems. The asculin band contains a compound that reacts with the asculin breakdown products and results in a color change. These bands are separated from one another by hydrophobic boundary layers. In use, a quantity of the β-hemolytic Streptococcus, the serological identification of which is to be determined, is placed on each of the three reagent bands. A drop of saline or water is added to the salicin area and to the esculin area. The strip is placed in a tube containing 0.3 ml of saline or water to wet the glycerin band. The strip impregnated with reagent is ^{incubated at approximately 37 °} C. for 2 to 4 hours. The sugar fermentation is measured by a change in the indicator color and the aculin hydrolysis by the formation of a gray-black color. the

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Group A Streptococci, which account for most of the clinical symptoms, like rheumatic fever and nephritis, are asulin-negative, glycerin-negative, and salicin-positive. Streptococci the Groups B and C can be found in diseases, but they are generally part of the normal flora in primary isolation of platelets showing ß-hemolysis. It is important to have one between these groups and group A. Can make distinction. The group D streptococci, known as enterococci, often occur when the urinary tract and rapid identification of these organisms is also clinically important.

Streptococci are gram-positive organisms that are round or oval in shape and have a tendency to form chains. These streptococci, which have primary pathogenicity to humans and animals, belong to the hemolytic subclass Streptococci. This subdivision is based on the observation that these streptococci cause red blood cell lysis, a property that is useful for the initial identification of these organisms in culture material. In a platelet which shows a clear zone, which is caused by the lysis of the red blood cells, the organism is called ß-hemolytic. Other types of hemolysis have also been described.

As a result of extensive serological studies, the ß-hemolytic streptococci were further transformed into a. number of divided serological groups, which differ and which in some way with the natural type of occurrence and the Pathogenicity of the organism are related. Serological identification was therefore useful in identifying pathogenic Streptococci an important step. It is in general known to be hemolytic streptococci, which are among the serological Groups A and D belong, not just for acute streptococca-Ie Diseases in humans are responsible, but that they also cause other diseases, such as rheumatic fever, nephrititis, -Bacteriuria and others. Accordingly ...... Ϊ098Γ8ΤΠ87Τ

it is important that the clinician know the serological group to which the organism belongs when having ß-hemolytic streptococci isolated so that appropriate treatment can be initiated. For example, if a doctor in a culture β-haemolytic streptococci from the upper respiratory tract discovered and he cannot type the isolated streptococci, he is often an antibiotic over a longer period of time Give time to avoid possible secondary diseases. In many cases when the organism is not a streptococci is group A, antibiotic treatment is not required. "..- ■ ··

In the serological groups as described by Lancefield (Journal of Experimental Medicine 57: 571-595, 1933), The identification is a function of the presence of certain groups of cellular "antigens. These antigens are." discovered by precipitate reactions between solutions of the extracts of the unknown Streptoeoeeus culture and antisera that were produced by immunizing rabbits with heat-killed suspensions of streptococci. It is easy to believe that such a procedure takes a lot of time and that one has to have staff available who is well trained.

A method has now been found according to the ß-hemolytic Streptococci can be easily identified into serological groups by a rapid and positive biochemical method. The present invention is explained in more detail with reference to the accompanying drawing.

Generally speaking, the preparation according to the invention contains a liner, which is typically a strip of a spongy material is limited to a wide variety Contains areas.

The surface 1 is impregnated with glycerine, a pH indicator system and optionally contains and possesses nutrient medium

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'! a pH value that, with reference to the pH indi- • kator is on the alkaline side.

; The face 2 is a hydrophobic boundary layer that is capable of

: the constituents must be prevented from migrating. Any

, Substance that forms a watertight barrier can be mixed with

j advantage to be used. Suitable materials include both

. for example waxes, lacquers and colorless acrylic resins,

I known as KRYLON 150 CRYSTAL CLEAR. This Krylon

I -

j material is particularly preferred. It's in a hydrocarbon ! Carrier agents are available and can be used for easier ani use with toluene or other hydrocarbon solvents,! for example with methyl, ethyl or isopropyl alcohol, verj be thinned.

The surface 3 is impregnated with salicin, a pH indicator system and optionally contains a nutrient medium and has a pH value that is on the alkaline side of the pH-In- ; indicator lies.

; The face 4 is a hydrophobic boundary layer that is the same is like that previously described for face 2.

ι The area 5 is. with Ä'sculin and an iron (III) salt, both

for example iron (III) chloride, iron (III) ammonium citrate, j iron (III) nitrite, iron (III) citrate or an aluminum salt, j for example aluminum ammonium sulfate, or a lead salt,! for example lead acetate or lead nitrate, impregnated.

I.
i
'■ The surface 6 is a hydrophobic boundary layer that is the same

; is as previously described for surface 2.

; The surface 7 is only optionally present and can be impregnated with any of the following: a hydrophobic material, as has been described above for the surface 2, and any suitable dye which is used for identification purposes. j ''

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The order of the reagent areas on the strip is not; critical as long as each surface is separated by a hydrophobic boundary layer but is the arrangement described; preferred. j

The indicator system for areas 1 and 3 contains indicators which result in a color change between a pH value of approximately pH 9 to approximately pH 5. Examples of these indicators are chlorophenol red, bromocresol purple, bromophenol red, bromothymol blue, neutral red, ^ -naphtholphthalein and phenol red. Examples of culture media are those that are commonly used to culture \ of bacteria, for example, peptone, tryptone, Proteose, tryptose, yeast extract, etc \

Sponge-shaped materials, under this term, are also intended. spongy and absorbent materials are understood that. Can be used as carriers are, for example Materials that draw a liquid upwards through capillary action, or materials such as filter paper, felt, porous ceramic strips, woven glass fibers or glass fibers in the form of mats and the like. ;

Alternatively, any absorbent material can be impregnated with the active ingredients and then on one Carriers, for example a plastic strip or a 'plastic strip, are attached. ;

For use, an eyelet full of organisms, which on one; ner agar plate with blood show ß-hemolysis, rubbed on the lower part of surface 1 and on surfaces 3 and 5. A drop of saline solution or water is also applied to areas 3 and 5 ^: . The resulting system is then placed in a test tube. with a size of 13 x 100 mm or a similar size, which contains 0.3 ml of saline solution, and then ^{incubated at 37 0} C for 2 to 4 hours.

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If phenol red is used as an indicator, a positive reaction in areas 1 and 3 is indicated by the formation of a yellow one Color displayed. A negative reaction is indicated by the remaining red color. The positive reaction is the result the fermentation of the sugars and the resulting acidity.

A positive reaction in area 5 is indicated by the development of a gray-black color, whereas a negative one Response is indicated by the absence of a color change. Again, the positive reaction is the result of the Fermentation of Ä'sculin and the conversion of the hydrolysis products with iron (III) salts or other compounds that are a Staining result.

By observing the presence or absence of the biochemical change, the serological identification of the ß-hemolytic streptococci can be obtained from the following table:

Streptococcus group A. B. G D. G Ä'sculin
Glycerin.
Salicin + +
d * +
d * +
+
d * -

! d * means variable

The advantages and usefulness of the present invention are apparent in terms of identifying of ß-hemolytic streptococci can easily occur within 2 to 4 hours.

Further group identification may be required through the use of other serological agents previously used in the experiments were, like the precipitin test, carried out

TQ9884 / TS73 "

The following examples illustrate the invention without, however, restricting it.

example 1

! Part A - Preparation of Glycerin Reagent Solution

Components

1. Glycerin USP

2. peptone

3. Phenol red

.4. distilled water qs 5. 5, On NaOH

for every 100 ml about 10-20g about 1-2g

200 mg

100 ml

qs

Ingredients 1, 2 and 3 are dissolved in 90 ml of distilled water dissolved with constant stirring. Ingredient No. is used to adjust the pH to 11.0. Then you add so much wasin - until - 100 ml of reagent solution is obtained.

geil B - production of salicin reagent solution

Components for 100 ml each

about 10-20g about 1-2g 200 mg
100 ml

Salicin Peptone Phenol Red Distilled Water qs

5. 5, On NaOH. qs

60 ml of distilled water were heated to 70 ⁰ C. To this, 1, 2 and 3 were added and dissolved with constant stirring. The pH was adjusted to 10.0 at 5 #. The volume with 4

• adjusted to 100 ml and then the mixture was heated to 7O ⁰ C.

Ί09 8 8 47187 3

I ■ - 8 -,

Part C - Preparation of esculin reagent solution ^: Ingredients * for 100 ml each

• 1. Esculin 2 - 5 g

2. Iron (III) ammonium citrate 1 - 2.5 g ι 3. Water qs

¹ 4. 1N sodium hydroxide solution qs

ι 80 ml of water was heated to 7O ⁰ C.. To this one added 1 and 2

■ and dissolved these compounds with constant stirring. The temperature was kept at 50 ^° C.-70 ^° C. The pH was adjusted to 4.3 ^{at 70 ° C. with j 4.} Since the esculin iron (III) -; If the ammonium citrate mixture is sensitive to light, the mixture should be protected from light.

I. ■ ■

Part-D - application to spongy material

ί To produce the test strips, you first set the boundary, surfaces 2 and 4, which are intended to prevent migration of the reagent solutions that are subsequently applied. These s Boundary zones are applied to a suitable support, preferably Eaton-Dikeman No. 623 filter paper, by using uses a lacquer solution that creates a water-impermeable layer results. After the lacquer solution has been allowed to dry, be. on surfaces 1, 3 and 5 the glycerol reagent solution, the j Salicin reagent solution and the esculin reagent solution, which according to prepared by the methods described above. The surfaces thus formed are allowed to dry. That Filter paper is then cut into strips.

Example 2

The following is an example of using the reagent strip it should be noted that there are many other uses.

T0988UT87

One or two loops full of streptococci that show ß-hemolysis on an agar plate with blood are on the lower side of the Surface 1 and ■ rubbed onto surfaces 3 and 5. The one made in this way The test system is placed in a 13 x 100 mm test tube that contains 0.3 ml of saline solution, so that area 1 is in the Saline solution is immersed. Incubate for 2 to 4 hours at 37 ° C. Identification of the serological groups to which belongs to the organism, takes place directly on the basis of the table mentioned above.

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Claims (8)

Claims 1. Diagnostic preparation for the identification of serological Groups of ß-hemolytic streptococci, characterized in that that it has a carrier "which contains: A) a first area covered with glycerine and an indicator system is impregnated that is able to withstand changes in pH between about 9 to about 5 display; , B) a second surface which contains a hydrophobic barrier layer and which is the first surface from the second surface separates; C.) a third area covered with salicin and a pH indicator is impregnated that is capable of indicating changes in pH from about 9 to 5; D) a fourth face, which is a hydrophobic boundary layer and which contains the third from the fifth surface: separates and E) a fifth surface covered with aesculin and a ferric salt or an aluminum salt or a lead salt is impregnated and F) a sixth face, which is a hydrophobic boundary layer contains S. 2. Diagnostic preparation according to claim 1, characterized in that ^: draws that the indicator system in the glycerine area j chlorophenol red or bromocresol purple or bromophenol red or j Bromthymol blue or neutral red or (X -naphtholphthalein 'or phenol red. . j-.
j ; 3. Diagnostic preparation according to claim 1, characterized ₉ that the indicator system in the salicinf pool Chloropheno red or bromocresol purple or bromophenol red or bromothymol blue or neutral red or crt -naphtholphthalein '■ or phenol red. i 4. Diagnostic preparation according to claim 1, characterized in that; indicates that the glycerin area and the salicin area also contain culture media. | 5. Diagnostic preparation according to claim 1, characterized in that that the iron (III) salt iron (III) chloride, iron (III) ammonium citrate, iron (III) nitrate or iron (III) citrate is. 6. Diagnostic preparation according to claim 1, characterized in that that the aluminum salt is aluminum ammonium citrate. 7. Diagnostic preparation according to claim 1, characterized in that that the lead salt is lead acetate or lead nitrate. 8. Method for the serological identification of ß-hänjciiylfischer Streptococci, characterized by the ^ -iflan organisms on the glycerine pool, the saline surface, and the aesculin surface the diagnostic preparation according to claim 1 in cubed, the enjskefiende system ^{incubated at 37 0} C for about 2 to 4> years and the color change of the surfaces> pay attention. 1-1 ^ * " August 4, 1971 Subject: Patent application P 21 29 584.5 Warner-Lambert Company Claim 8? · & 'M Use of the diagnostic preparation according to claim 1 for the serological identification of ß-hemolytic streptococci, organisms being incubated on the glycerol surface, HWt the salicin surface and the esculin surface of the diagnostic preparation, the resulting system at yj%. ™ Incubated for 2-4 hours and observed the color change of the colored areas. 109884/1873