WO1991006867A1

WO1991006867A1 - Echinoderm agglutinin and method of extraction

Info

Publication number: WO1991006867A1
Application number: PCT/CA1990/000371
Authority: WO
Inventors: Alastair Connell Wardlaw; Charles Maxwell Moseley
Original assignee: Atlantic Institute Of Biotechnology
Priority date: 1989-11-01
Filing date: 1990-10-29
Publication date: 1991-05-16
Also published as: EP0498820A1; AU6613790A; CA2002000A1; JPH06504112A

Abstract

A method is disclosed for extracting an endotoxin-detecting agglutinin from the coelomic fluid of Echinoderms of the Subclass Regularia. The product is useful in detecting Gram-negative bacteria, endotoxins and endotoxin contamination of injectable and intravenous fluid and medical devices. The product is an alternative to the expensive Limulus Amoebocyte Lysate reagent.

Description

ECHINODERM AGGLUTININ AND METHOD OF EXTRACTION

The present invention relates to Echinoderm agglutinins and a method for extracting them from the organisms.

Endotoxins are a serious problem in medicine. These pharmacologically active chemicals are produced primarily by Gram-negative bacteria. They are lipopolysaccharides (LPS) from the bacterial cell membrane. The toxins are ubiquitous, heat stable and active, even in picogram amounts (ie IO^"12 g) . Endotoxins can contaminate injectable fluids, intravenous solutions and medical devices such as fluid lines, syringes and blood bags.

No amount of cleanliness can preclude endotoxin contamination in the manufacture of drugs and infusion fluids. Ordinary tap water has detectable amounts of endotoxin, even when it has been autoclaved. Heating will not detoxify the contaminant. The water must be purified, and re-contamination is also possible. Regular endotoxin testing is required.

Endotoxin can cause fever when injected. A dose as low as 0.1 ng/kg produces fever which can be fatal in a debilitated patient. In higher doses, endotoxin causes shock and can be lethal in more healthy people.

Pharmaceutical and medical device companies must keep endotoxins from contaminating their products.

Until the late 1970's, the only means for testing endotoxins was the United States Pharmacopeia (USP) rabbit pyrogen test, first developed during World War II. Rabbits are injected with intravenous or injectable fluids, or alternatively with fluid from the flushing of medical devices with non-pyrogenic saline solution. Temperatures are then taken for 3 hours. A significant rise in rabbit temperature indicates presence of pyrogenic endotoxin. Rabbits are used because their response to endotoxin most closely resembles that of man. The test has many drawbacks. It is subject to variation and false results. Rabbit colonies are expensive to maintain, and routine testing with animals is becoming increasingly socially unacceptable.

In 1964, J. Levin and F. Bang showed, in Bulletin of Johns Hopkins Hospital, 115: 265-274, that bacterial endotoxin triggered a clotting reaction in the blood of the horseshoe crab, Limulus polyphemus. Picogram quantities of endotoxin cause Limulus amoebocyte blood lysate to form a proteinaceous gel. The reaction seems to be a primitive clotting of Limulus blood.

The Limulus Amoebocyte Lysate (LAL) endotoxin test is simple, rapid and more sensitive than the rabbit pyrogen test. LAL is also cheaper, and requires no special facilities. The LAL test was allowed by FDA in the United States as a substitute for the rabit pyrogen test for biologies.

The LAL test does have some problems. The horseshoe crab is not common. There are not enough animals to satisfy the enormous demand. LAL reagent is expensive, and requires extensive skilled labor to extract it.

An alternate, more abundant source for endotoxin agglutinins would be desirable.

A few invertebrates have been examined for agglutinin activity in the twenty-five years since the Limulus LAL discovery. K. SOderhall et al. discovered and claimed agglutinin behavior in crustaceans, such as lobsters, and insects, in P.C.T. application WO 83-02123.

It has been discovered that echinoderms, of the sub¬ class Regularia have coelomocytes that agglutinate when contacted with endotoxin extracted from a number of gram- negative bacteria.

The echinoderms are plentiful in the Atlantic and Pacific oceans. Most of them presently have no commercial application, and some of them are pests. Sea urchins of the genus Strongylocentrotus damage the commercially-harvested Macrocvstis (Giant Kelp) beds along the California coastline, because they graze on the plants, and the Green Sea Urchin, Strongylocentrotusdroebachiensis can overgrazeLaminariakelps off Atlantic Canada thereby reducing catches of lobsters.

The invention seeks to provide a new agglutinin reagent for the testing of endotoxin. The invention further seeks to provide an alternate, potentially cheaper source for an endotoxin agglutination and testing reagent.

Accordingly, one aspect of the invention provides for a method of manufacturing an endotoxin agglutinin reagent, which comprises extracting whole coelomic fluid from the coelome of an echinoderm animal of the sub-class Regularia.

Another aspect of the invention provides for the further steps of cleaning the animal*s peristomial membrane surface, extracting coelomic fluid using needle means, and conducting dilutions, where required, using endotoxin-free sterile saline fluid of 3.4% sodium chloride (weight by volume) .

The invention further seeks to provide a reagent for detecting endotoxin comprising the whole coelomic fluid of an echinoderm animal of the sub-class Regularia.

The following echinoderms showed a positive agglutination response when their coelomic fluid was contacted with endotoxin.¹

TABLE 1

ORGANISM SOURCE

CLASS: ECHINOIDEA* SUBCLASS: REGULARIA

Order: Cidaroidea Family: Cidaridae

Eucidaris sp. Hawaii

Order: Aulodonta

Family: Diadematidae

Diadema multispinum Hawaii

Echinothrix calamaris Hawaii

¹ E. coli Serotype No. 055:B5 phenol extract, Sigma catalog . L-2880 Order: Stirodonta

Family: Arbaciidae Arbacia punctulata Woods Hole,

USA

Order: Camarodonta

Fa ily: Toxopneustidae Tripneustes qratilla Hawaii

Family: Echinidae Echinus esculentus Scotland Psammechinus miliaris Scotland

Family: Strongylocentrotidae

Strongylocentrotus droebachiensis Nova Scotia Strongylocentrotus droebachiensis B r i t i s h

Columbia

S. franciscanus B r i t i sh

Columbia

S . purpuratus B r i i sh

Columbia

Family: Echinometridae Echinometra mathaei Hawaii Heterocentrotus mammillatus Hawaii Colobocentrotus atratus Hawaii

* Classifications follow those of Libbie Hyman in "The invertebrates:Echinodermata." Vol 5, McGraw-Hill 1955.

Some members of the same class, but of the sub-class Irregularia do not show agglutination with the same endotoxin. These organisms are:

TABLE 2

ORGANISM SOURCE

CLASS: ECHINOIDEA SUBCLASS: IRREGULARIA

Order: Clypeastroida ("Sand Dollars") Family: Scutellidae Echinarachnius parma Nova Scotia

Dendraster excentricus British Columbia

Order: Spatangoida ("Heart Urchins") Family: Loveniidae

Echinocardium cordatu Scotland

These echinoderms are known collectively as sand dollars and heart urchins.

12. coli endotoxin is not the only endotoxin that these organisms agglutinate. A series of endotoxins from Gram- negative bacteria was tested with coelomic fluid from the green sea urchin Strongylocentrotus droebachiensis. Agglutination was seen with the following endotoxins:

TABLE 3

1. Escherichia coli Serotype No. 055:B5. Phenol extract. Sigma Catalogue No. L-02880. Lot # 17F-40191.

2. E. coli Serotype 0127:B8. Phenol Extract. Sigma Cat. No. L-3129. Lot # 47F-4046.

3. E. coli Serotype 0111:B4. Phenol extract. Sigma Cat. No. L-2630. Lot # 97F-4089.

4. Salmonellaminnesota R7 (Rdmutant) Phenol-Chloroform- Petroleum ether extract. Sigma Cat. No. L-9391. Lot # 96F-4016.

5. Salmonella abortus-egui. Phenol extract. Sigma Cat. NO. L-5886. Lot' # 104F-4027.

6. Pseudomonas aeruginosa. Phenol extract. Sigma Cat. No. L-9143. Lot # 87F-4009.

7. Serratia marcescens. Phenol extract. Sigma Cat. No. L-6136. Lot # 93F-4019.

Two other preparations of micro-organisms were also tested. Neither sample caused the green sea urchin coelomic fluid to undergo agglutination. The two organisms were:

1. a preparation of brewers¹ yeast, the fungus Saσcharomyces cerevisiae. that was first boiled;

2. a boiled and cooled preparation of Micrococcus luteus. a Gram-positive bacterium.

Neither of these organisms produces endotoxins.

The echinoderm agglutinin of the present invention is very sensitive. Fresh coelomic fluid of the green sea urchin was tested against serial dilutions of E. coli endotoxin, Serotype 055:B5, phenol extract. Table 4 shows the results.

TABLE 4

Concentration of Agglutination response endotoxin (ng/ml)

400 Strong agglutination and cl

200 formation

100

50

25

12.5 Weak agglutination with parti

6.25 resuspension upon light shakin

3.125 No visible agglutination coelomocytes

Another experiment showedagglutinationwith endotoxin concentrations as low as 2.5 ng/ml, as reflected in Table 6, Example 3.

Sea urchins, though not closely biologically related, are similar in form to horseshoe crabs. They are both relatively large, hard-shelled marine invertebrates of ancient lineage. Both organisms have a relatively long lifespan measured in decades, and live on the sea floor, where bacteria are abundant and are liable to invade the body cavity of the animal after mechanical injury.

Both animals exhibit clotting capability, but the sea urchin coelomic (body) fluid differs markedly from the blood of the horseshoe crab. Horseshoe crab fluid is protein-rich and has a single cell type called an amoebocyte. This cell type is functionally similar to the mammalian platelet. In contrast, the sea urchin coelomic fluid is essentially protein- free, and contains several types of cells, collectively termed coelomocytes. These cell types may be separated by density gradient centrifugation; their precise function or functions are unknown, excepting that they are involved in clotting. The coelomic fluid of some sea urchins is anti-bacterial.

The echinoderms which make endotoxin agglutinins may not make them year-round. There is some evidence that these animals digest their own cells during periods of starvation. The coelomocyte cells are metabolized early on in this process. There may be other biological reasons for the disappearance of coelomocyte cells, or for their failure to respond to introduced endotoxin.

In light of this observation, experiments were conducted to further study the agglutination response. Seasonal tests were done on selected temperate zone echinoderm species from the North American east and west coast, and from Europe.

Table five shows results of sampling for agglutinin activity over three years. E. coli endotoxin was used. TABLE 5 Agglutination Response Species Month (1987-1989)

O N D

* North America - East coast ** North America - West coast *** Europe

Mean ambient seawater temperatures in 1989, typical of water temperatures for those months, are as follows ( C) :

July 13.7 August 11.2 October 10.9

A +/- means a questionable agglutination. A negative agglutination of coelomocyte cells and endotoxin does not necessarily mean that the organism cannot agglutinate endotoxin. The animal may be able to do so at another time of year or under different culture conditions.

In accordance with the method of the invention, an echinoderm agglutinin extract may be made in the following manner.

Echinoderms such as sea urchins are collected from the ocean by researchers using scuba equipment. The animals are transported to aquarium tanks, kept at about 10 C.

The animal is detached from the wall of the aquarium tank. A rapid approach and grab technique is used. This method does not give the animal enough time to strengthen its hold through its tube feet. Care should be taken not to rip the tube feet from the animal. Wounding and the subsequent contamination should be avoided. Animals that have a firm hold on the aquarium wall should be by-passed until they become relaxed again.

The animal is then placed on a paper towel, mouth side uppermost. The peristomial membrane on the oral area is cleaned thoroughly with sterile cotton swabs on sticks. The swabbing is done in a continuous circular motion with rotation of the swab. Several swabs are used in succession until very little or no brown material appears on the swab. The swabbing may be done up to six times. The peristomial area may be cleaned further by rinsing with a small amount of endotoxin- free 3.2% saline between swabbings. Alternatively, 95% ethyl alcohol may be used for the swabbing step. This cleaning removes the surface slime and endotoxin from the peristome surface and in the seawater thereon.

Coelomic fluid sampling needles should be sterile and pyrogen free. Plastic disposable syringes are usually used. Suitable sizes are 1 ml, 5 ml and 10 ml. The latter two sizes are preferred. Syringes, in their original wrappers, are cooled by burial in crushed ice for at least 10 minutes before use.

In coelomic fluid sampling, the animal is held oral side downwards, and above eye level.

The syringe needle is inserted to about two-thirds of its length. Puncture is made at a point close to the outer margin of the peristomial membrane, and at an angle of thirty degrees from the oral-aboral axis. The plunger is withdrawn with a steady force to fill the syringe. Excessive suction is avoided, as it produces bubbles. Whole coelomic fluid is extracted. This whole fluid comprises fluid and coelomocytes. The amount of harvested coelomic fluid from each animal is considerable. For example, an average-sized green sea urchin (about 6 cm test diameter) yields about 10 ml. The animal can survive the loss if it is put into a recovery tank.

The removed whole coelomic fluid is dispensed directly and without delay to test tubes containing desired reagents such as bacterial lipopolysaccharide (endotoxin) or endotoxin- free saline solution. These test tubes are place on ice before filling. Fluid dispensing is done slowly, to avoid damage to the cells.

All glassware that is used in working with coelomic fluid is specially treated to destroy all endotoxin. For example, glass test tubes are capped with aluminum foil and baked in an oven at 200 C for at least 4 hours to render them endotoxin- free.

Endotoxin-free saline only is used for diluting the coelomic fluid, where required. The saline solution is produced by first placing weighed portions of sodium chloride in dry glass tubes that are covered by aluminum foil. The tubes are baked at 200 C for four hours to destroy any endotoxin. The saline solution is made up by placing sterile endotoxin-free water into those tubes. The endotoxin-free water can be purchased from hospital supply companies.

Suitable quantities are, for example, 0.34 grams of NaCl in a 16 by 100 mm glass tube. 10 ml of water may then be added to give a 3.4% (w/v) saline solution which is approximately iso-osmotic with sea urchin coelomic fluid.

The endotoxin test solution was prepared by preparing a 5 mg/ml stock solution of purchased endotoxin in a 3.4% weight by volume NaCl solution. This solution is sterilized by boiling the solution on three successive days for ten minutes each time. The stock solution may be serially diluted with added 3.4% sterile, pyrogen-free NaCl solution. A fresh syringe is used at each successive transfer, to help forestall contamination and unwanted agglutination. The endotoxin most commonly used for agglutination experiments was E. coli Serotype 055:B5, purchased from Sigma Laboratories.

Several anti-coagulant chemicals were also investigated. These chemicals can stabilize agglutinin systems while they are extracted but allow the agglutination reaction to occur later, when desired.

Three reagents were tested, but none proved satisfactory. The accepted Limulus anti-coagulant N- ethymaleimide did not work at all. Similarly, the mixture of P-toluenesulfonyl-arginine-methyl ester (TAME) , mercaptoethanol and caffeine did not stop clotting in the syringe. The third anti-coagulant, EGTA, prevented clotting, but the agglutinin would not react with endotoxin. There is a possibility that less vigorous chelating agents may prove suitable.

The following are potential specific applications for agglutinins of the invention;

(a) The agglutinins recognize endotoxins and subsequently may be used for the detection of Gram- negative bacteria or their free lipopolysaccharide endotoxins. Injection fluid, sera, and intravenous fluid, for example, may be tested. Medical devices used for injections, taking samples, intravenous feeding or drug delivery, etcetera, may be flushed and the fluid checked for contamination.

(b) The agglutinins may be used for affinity purification of lipopolysaccharides (LPS) from most Gram-negative bacteria. Such LPS may be used in scientific research and for medical purposes. For example, the LPS antigenicity or serotype can help idenify a Gram-negative bacterium. Additionally, such LPS purification could result in very pure antigens to be used in monoclonal antibody screening for diagnostic purposes. Such LPS antigens may also be used in the preparation of ELISA test reagents and sandwich antibody diagnostics.

(c) The agglutinins may be used to remove LPS from injectable drug solutions, sera and vaccine preparations.

(d) Agglutinin may be used to test for endotoxin contamination in the environment, water, food, etc.

(e) Other uses can be readily discovered by those skilled in the art, and the spirit of the invention should not be restricted to any one of them.

The following Examples illustrate the invention.

EXAMPLE 1

Green sea urchins (Srongylocentrotus droebachiensis) were collected by SCUBA divers off Sandy Cove, on the Fundy Shore of Nova Scotia, Canada. The sea urchins were transported to aquarium tanks and kept at 10 C.

Just before the experiment, specimens were removed from the aquarium wall. This was done by a rapid approach and grab, to avoid having the animal become tense and strengthen its hold through its tube feet. Ripping of tube feet should be avoided.

Each animal was then placed mouth-side uppermost on a paper towel, and the area of the peristome cleaned thoroughly with sterile cotton swabs on sticks. The swabbing was done in a continuous circular motion with rotation of the swab, and several swabs were used in succession until no more, or very little, brown material appeared on the swab. For further cleansing, the peristome was rinsed with a small quantity of endotoxin-free saline between swabbings.

Meanwhile, 5 ml or 10 ml syringes with 21 gauge 1.5 inch needles, still in their wrappers, had been cooled by burial in crushed ice for at least 10 minutes beforehand.

To take coelomic fluid, the sea urchin was picked up in the left hand, and held oral side downwards, above the operator's eye level. The syringe needle was inserted, to about two-thirds of its length, at a point close to the outer margin of the peristomial membrane and at an angle of about 30 away from the oral-aboral axis. The plunger was withdrawn with steady force to fill the syringe, but without creating bubbles by excessive suction. An average-sized green sea urchin (6 cm test diameter) , yielded about 10 ml coelomic fluid, and can survive this loss if put in a recovery tank.

The coelomic fluid was dispensed directly and without delay from the syringe into tubes containing LPS or endotoxin- free saline.

The fluid was slowly taken up by the syringe to avoid shearing force. Similarly, fluid was carefully dispensed into tubes by contacting the inner tube wall, to avoid frothing.

Twenty sea urchins were used. Anticoagulant-free whole coelomic fluid was collected, and 500 microlitre aliquots were mixed with one of: (a) 10 microlitres of 3.2% saline, (b) 0.5 micrograms of E. coli endotoxin, or (c) 50 micrograms of E. coli endotoxin.

In coelomic fluid from 17 of the sea urchins, "clots" formed in the tubes which contained endotoxin (both concentrations) . No clot was formed in the tubes that had no endotoxin. The fluid from the other 3 sea urchins gave positive clot reactions in all three tubes. These three samples were probably contaminated. This experiment showed that careful swabbing of the organism and withdrawal of fluid could prevent premature clotting.

EXAMPLE 2

Coelomic fluid was withdrawn as described in Example 1. The organism was the green sea urchin. 0.5 ml portions of coelomic fluid were added to tubes containing saline and either microgram or nanogram quantities of endotoxin, or 0.05 ml of endotoxin-free saline (tested in duplicate) . The tubes were left to react undisturbed for 30 minutes at 20 C. The following table shows the results of the coelomic fluid with diminishing concentration of LPS-endotoxin.

TABLE 6

Response

No clot formed. Coelomic cells settled onto the bottom of the

A clot formed which looked like

inverted cone from the meniscus, with a bulb at the base.

3 2.5 micrograms A large spheroid clot formed

4 0.25 micrograms Same response as tube 3

5 25 nanograms Same response as tube 2

6 2.5 nanograms A clot formed on the tube base

7 0.0 Same response as tube 1

The lowest concentration, 2.5 nanograms, showed a vigorous clotting response of coelomic fluid. The clot was bulky. It is conceivable that there will be a clotting response at 0.25 ng. This brings the echinoid clotting response into the range of the LAL sensitivity in its formative years.

EXAMPLE 3

Coelomic fluid was extracted from a variety of Echinoid species with the procedure described in Example 1. 5 ml of fresh coelomic fluid of each organism was added to a solution of endotoxin from Escherichia coli Serotype 055:B5, phenol extract, Sigma catalog No. L-2280. Tubes were examined for agglutination (clotting) . Those tubes which appeared to have a clot were gently tapped to see if they would resuspend. If the cells did resuspend upon tapping, then they had merely settled and not clotted, and the result was deemed to be a non-clotting response. The results are reported in Tables 1 and 2 of the disclosure.

EXAMPLE 4

The green sea urchin, Strongylocentrotus droebachiensis . was used to test the effect of whole coelomic fluid with an assortment of endotoxins. These endotoxins were derived from different Gram-negative bacterial organisms.

Whole coelomic fluid was extracted using the procedure described in Example 1. All endotoxin samples were standardized at 2400 EU per ml. Sterile pyrogen-free water was used throughout. The test endotoxin solutions were each added to 0.5 ml of the sea urchin whole coelomic fluid. All of the endotoxin samples showed agglutination, and all the controls were negative (ie. no agglutination) . Results of this experiment are given in Table 3 of the disclosure.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE AS FOLLOWS;

1. A method for manufacturing an endotoxin agglutinin reagent which comprises extracting whole coelomic fluid from the coelome of an echinoderm of the sub-class Regularia.

2. A method according to claim 1, wherein the animal's peristomial membrane surface is cleaned and the whole coelomic fluid is extracted using needle means.

3. A method according to claim 2 wherein said whole coelomic fluid is diluted using endotoxin-free sterile saline.

4. A method according to claim 3 wherein said saline is 3.4 % weight by volume sodium chloride in endotoxin-free sterile distilled water.

5. A method according to claim 1, 2 or 4 wherein said echinoderm is selected from the group comprising the orders Cidaroidea, Aulodonta, Stirodonta or Camarodonta.

6. A method according to claim 1, 2 or 4 wherein said echinoderm is selected from the group comprising the families Cidaridae, Diadematidae, Arbaciidae, Toxopneustidae, Echinidae, Strongylocentrotidae, Echinometridae.

7. A method according to claim 1, 2 or 4 wherein said echinoderm is selected from the group comprising the genera Eucidaris. Diadema, Echinothrix_f Arbacia. Tripneustes. Echinus. Psa mechinus. Strongylocentrotus. Echinometra. Heterocentrotus. Colobocentrotus.

8. A method according to claim 1, 2 or 4 wherein said echinoderm is selected from the group comprising the species Diadema multispinum Echinothrix calamaris Arbacia punctulata Tripneustes gratilla Echinus esculentus Psammechinus miliaris Strongylocentrotus droebachiensis S. franciscanus ϋ« purpuratus Echinometra mathaei Heterocentrotus mammillatus Colobocentrotus atratus

9. A reagent for detecting endotoxin comprising the whole coleomic fluid of an echinoderm animal of the sub-class Regularia.

10. A reagent according to claim 9 wherein said echinoderm animal is selected from the group comprising the orders Cidaroidea, Aulodonta, Stirodonta or Camarodonta.

11. A reagent according to claim 9 wherein said echinoderm animal is selected from the group comprising the families Cidaridae, Diadematidae, Arbaciidae, Toxopneustidae, Echinidae, Strongylocentrotidae, Echinometridae.

12. A reagent according to claim 9 wherein said echinoderm animal is selected from the group comprising the genera Eucidaris, Diadema. Echinothri . Arbacia. Tripneustes. Echinus, Psammechinus. Strongylocentrotus. Echinometra. Heterocentrotus, Colobocentrotus.

13. A reagent according to claim 9 wherein said echinoderm animal is selected from the group comprising the species

Diadema multispinum Echinothrix calamaris Arbacia punctulata Tripneustes gratilla Echinus esculentus Psammechinus miliaris Strongylocentrotus droebachiensis s. franciscanus S. purpuratus Echinometra mathaei Heterocentrotus mammillatus Co1obocentrotus atratus

14. A reagent for detecting endotoxin comprising coelomic fluid and coelomocytes of Strongylocentrotus droebachiensis.

15. A reagent according to claims 9, 10 or 11 characterized by the ability to agglutinate any endotoxins of the group comprising;

Escherichia coli Serotype No. 055:135. Phenol extract. Sigma Catalogue No. L-2880.

E. coli Serotype 0127:B8. Phenol Extract. Sigma Cat. No. L-3129.

E. coli Serotype 0111:B4. Phenol extract. Sigma Cat. No. L-2630.

Salmonella minnesota R7 (Rd mutant) Phenol-Chloroform- Petroleum ether extract. Sigma Cat. No. L-9391.

Salmonella abortus-equi. Phenol extract. Sigma Cat. No. L-5886.

Pseudomonas aeruginosa. Phenol extract. Sigma Cat. No. L- 9143.

Serratia marcescens. Phenol extract. Sigma Cat. No. L-6136.

16. A reagent according to claims 12, 13 or 14 characterized by the ability to agglutinate any endotoxins of the group comprising;

Escherichia coli Serotype No. 055:B5. Phenol extract. Sigma Catalogue No. L-2880.

E. coli Serotype 0127:B8. Phenol Extract. Sigma Cat. No. L-3129.

3ϋ« coli Serotype 0111:B4. Phenol extract. Sigma Cat. No. L-2630.

Salmonella abortus-equi. Phenol extract. Sigma Cat. No. L-5886.

Pseudomonas aeruginosa. Phenol extract. Sigma Cat. No. L- 9143.

Serratia marcescens. Phenol extract. Sigma Cat. No. L-6136.