Microgen Bacillus ID is a miniaturized biochemical identification system designed to identify those Mesophilic Bacillus spp. and related genera associated with food and beverage spoilage and food poisoning. This identification system comprises 24 biochemical substrates SPECIFICALLY selected and formulated to provide accurate and efficient identification of Bacillus spp.

This product saves time and labor, while improving the accuracy and cost of identification of these organisms. The Microgen™ Bacillus ID employs the most up-to-date taxonomy. Each kit contains suspending medium and sufficient microwell strips for 20 identifications, holding tray, instructions, and reporting cards. Reagents and Identification Software are available separately.

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Features:

  • Small 24-well test system
  • Simple, easy to use and read
  • Final result in 48 hours
  • Substrates specific for Bacillus
  • Based on conventional methods
  • Identifies all common species found in food and related samples
  • Includes CARBOHYDRATE CONTROL well as reference for color interpretation
  • Up-to-date taxonomy

Benefits:

  • Confidence in results
  • Quick to inoculate and simple to read
  • Timely results
  • Accuracy and security of reporting correct name
  • Reduces errors
  • Can cross reference with standard texts
  • Cost effective

Easy to use and read:

  • Select sufficient colonies from a purity plate
  • Emulsify colonies in the suspending medium provided, equivalent to a MacFarland 2
  • Inoculate each well of the strips
  • Incubate 24 hours at 30°C
  • Read and record carbohydrate, ONPG and citrate reactions
  • Incubate further 24 hours at 30°C
  • Add reagents then re-read strips and record results
  • Analyze code with the Microgen Identification System Software

Bacillus Information:

  • Clinical contaminationincludes operating rooms surgical dressings, and in pharmaceutical products.
  • Food Spoilage:Bacillus spp. can be isolated from fruits, vegetable products, nuts, spices, cereals, milk and dairy products, meat and dried foods.
  • Food Poisoning:Isolates of B.cereus, B.brevis, B.licheniformis, B.subtillis, and B.sphaericus have been found
  • Milk and Milk Products:B.licheniformis is a common contaminant of dairy products. Paenibacillus spp., Brevibacillus spp. and Virgibacillus spp. are implicated in spoilage.

Species identified using the Microgen™ ID Bacillus

Bacillus species

  • B. cereus group
  • B. firmus
  • B. badius
  • B. laevolacticus
  • B. coagulans
  • B. lentus
  • B. amyloliquefaciens
  • B. subtilis
  • B. circulans
  • B. insolitus
  • B. freudenreichii
  • B. globisporus
  • B. sphaericus
  • B. thiaminolyticus
  • B. pumilus
  • B. licheniformis
  • B. megaterium

Vergibacillus species

  • V. pantothenticus

Paenibacillus species

  • P. polymyxa
  • P. macerans
  • P. alvei

Brevibacillus species

  • Br. brevis
  • Br. laterosporus

Quick Guide to using the Microgen™ ID Bacillus ID Kit

Step 1

Prior to the identification of any isolate it must be confirmed as belonging to the genus Bacillus.

  1. Gram positive bacillus
  2. Endospore forming
  3. Catalase positive
  4. Optimal growth temperature between 25 and 45°C i.e. Mesophilic. Isolates growing at < 25°C (Psychrophiles) or isolates growing at 45°C (Thermophiles) are not identified by this product.
Step 2

Prepare a suspension equivalent to MacFarland No. 2 in the suspending medium provided. As some colonies may be very dry and difficult to emulsify to produce a homogeneous suspension it is recommended that a moist sterile swab be used to harvest colonies and re suspend. Additional mixing using a vortex mixer may be necessary. When using a swab to harvest growth, this should be done from a purity plate to ensure that mixed cultures are not created.

Step 3

Peel back the adhesive sealing tape from each strip. Inoculate the microwells in both test strips with 3 drops (approx 100µl) of inoculum.

Step 4

Overlay the Arginine well with Mineral oil.

Step 5

Seal the strips. Incubate at 30ºC for 18 – 24 hours.

Step 6

Read and record the results of all Sugar Fermentation tests PLUS the Citrate and Urease tests. Incubate at 30ºC for a further 18 – 24 hours.

Step 7

Re read the results of all Sugar Fermentation tests. Add reagents (Indole, Nitrate, VP I & II).

NOTE: Occasionally some sugar fermentation tests may be positive after 18– 24 hours incubation but negative after 48 hours incubation. When the final Octal Code is calculated, all tests which showed a positive reaction after either 18 – 24 hours incubation or 48 hours incubation should be scored as POSITIVE. Only reactions negative at both readings should be scored as NEGATIVE.

Step 8

Record all results on report form provided. Sum the accumulated positive test results from both the 24 and 48 hour readings.

Step 9

Analyze the results using Microgen Identification System Software

bacillus_id_results_form

This product saves time and labor, while improving the accuracy and cost of identification of these organisms. Each kit contains suspending medium and sufficient microwell strips for 20 identifications. Reagents and Identification Software are available separately. For further information, please contact Microbiology International at (800)396-4276.

For a complete explanation of procedure for use, please refer to the Instructions for Use, included with your kit.

Background Information

The genus Bacillus represents a large and diverse group of species including B. cereus which is well known as a causative agent of food poisoning. However, many other species of Bacillus and related genera have over the years also been implicated in outbreaks of food poisoning. Originally only 16 species of Bacillus were recognized. However, over the years additional species have been recognized and with the introduction of molecular methods, the genus Bacillus now includes 123 recognized species plus an additional 7 genera comprising closely related organisms are now recognized. Because these organisms are so widely distributed in the environment they are able to easily contaminate food ingredients and products. In addition, many of these organisms have been demonstrated to produce toxins capable of causing both diarrhea and vomiting. It is for this reason that the Microgen Bacillus ID includes not only some of the mesophilic Bacillus spp. but also some species belonging to Vergibacillus, Brevibacillus and Paenibacillus. All of the species included in this identification system have been isolated and/ or reported as causing food spoilage or food poisoning.

Bacillus species and food poisoning

Although B. cereus is the most common species of Bacillus associated with food poisoning, a range of other species have been implicated in foodborne illnesses. The two most commonly reported species are B. subtilis and B. licheniformis, these species being of concern to the food processing and canning industries as they are often implicated as causes of food spoilage producing potent proteolytic and saccarolytic enzymes. Some strains of B. licheniformis have been shown to produce toxins. In addition, a range of other species including B. pumilus, B. brevis and B. thuringiensis, have also been reported as being the sole species implicated in outbreaks of food poisoning. B. lentus, B. circulans, B. mycoides, B. polymyxa and B. laterosporus have also been demonstrated to produce toxins using cell cytotoxicity assays whilst some of these species have also been shown to produce positive RPLA and TECRA™ assays.

B. subtilis is the most frequently reported species associated with food poisoning other than B. cereus , being associated with outbreaks involving meat, pastry products, seafood and rice. Incubation periods varied from 1 – 4 hours, with recovery generally complete within 24 hours. Symptoms usually included nausea and vomiting, diarrhea was rarely reported. B. licheniformis associated food poisoning resembles that of C. perfringens, with an incubation period of 6 – 24 hours and symptoms including diarrhea and abdominal pain. In cases of food poisoning associated with B. licheniformis, high numbers of the bacteria 17 (106 cfu/gm) were found in the implicated food (often cooked meat and vegetable dishes) and in the feces of affected persons.

B. pumilus was implicated in 5 outbreaks of food poisoning between 1975 and 1986 in the UK , in each of these cases only a small number of individuals were involved, however in each case high numbers > 6 x 106 organisms/ gm were isolated from the implicated foods in the absence of other recognized foodborne pathogens. Gilbert et. al. reported 4 instances of food poisoning in which B. brevis was the sole organism isolated in high numbers from foods implicated in food poisoning outbreaks. In any investigation of suspected food poisoning in which none of the normal primary pathogens such as Salmonella, Campylobacter, E. coli, C. perfringens and S. aureus are isolated Bacillus should always be considered as a potential cause. In such studies, particularly if the symptoms exhibited by patients include diarrhea, species other than B. cereus should always be considered.

Technical Information for Microgen™ Bacillus ID
Storage: The microwell strips are stable in the unopened foil pouches at 2-8°C until the expiry date stated. The Bacillus suspending broth should be stored at 2-8°C.
Shelf Life: 1 year

Species Identified

Bacillus species

  • B. cereus group
  • B. firmus
  • B. badius
  • B. laevolacticus
  • B. coagulans
  • B. lentus
  • B. amyloliquefaciens
  • B. subtilis
  • B. circulans
  • B. insolitus
  • B. freudenreichii
  • B. globisporus
  • B. sphaericus
  • B. thiaminolyticus
  • B. pumilus
  • B. licheniformis
  • B. megaterium

Vergibacillus species

  • V. pantothenticus

Paenibacillus species

  • P. polymyxa
  • P. macerans
  • P. alvei

Brevibacillus species

  • Br. brevis
  • Br. laterosporus

Well Substrates

  • Well 1- Arabinose
  • Well 2- Cellobiose
  • Well 3- Inositol
  • Well 4- Mannitol
  • Well 5- Mannose
  • Well 6- Raffinose
  • Well 7- Rhamnose
  • Well 8- Salicin
  • Well 9- Sorbitol
  • Well 10- Sucrose
  • Well 11- Trehalose
  • Well 12- Xylose
  • Well 13- Adonitol
  • Well 14- Galactose
  • Well 15- Methyl-D-Mannoside
  • Well 16- Methyl-D-Glucoside
  • Well 17- Inulin
  • Well 18- Melizitose
  • Well 19- Indole
  • Well 20- ONPG
  • Well 21- Arginine
  • Well 22- Citrate
  • Well 23- VP
  • Well 24- Nitrate

Reagents

  • Indole Kovacs
  • Voges-Proskauer I
  • Voges-Proskauer II
  • Nitrate Reagent A
  • Nitrate Reagent B
  • Mineral Oil