Isolation of Bacillus thuringiensis (Bt) from Soil sample
Isolation of Bacillus thuringiensis (Bt) from Soil sample
A common, Gram-positive, and sporulating bacterium called Bacillus thuringiensis (Bt) produces insecticidal proteins with selectivity for a variety of insects during sporulation (Cry and Cyt) and vegetative growth (Vip and Sip). These proteins have promoted Bt as a more sustainable pesticide than chemical ones.
Principle of Bacillus thuringiensis Isolation
Thermal shock treatment followed by selective spore germination are the methods that are frequently used to separate Bt from soil. All bacteria unable to produce endospores are eradicated from the sample soil using thermal shock. After that, the samples are multiplied many times to lower the amount of colony-forming units and to remove the humic substances from the soil. In order to give the spores an opportunity to germinate on medium with enough nutrients and at the right temperature, the samples are then grown on nutritional agar. A wide variety of microorganisms, including Bacillus thuringiensis, can grow well in the media. Thus, further selection procedures are used to isolate Bacillus thuringiensis from the diverse population of microorganisms present in the basic soil sample. Common and significant tests include Gram’s staining, Amino black and Ziehl’s carbol fuchsin staining, Endospore staining, Catalase test, determining whether growth occurs above 45 °C, and determining whether parasporal bodies are present, among others.
The steps involved in isolating Bacillus thuringiensis
- After removing the top 2-3 cm, collect around 20 g of cultivated or uncultivated soil with a tubular soil sampler.
- Until isolation, place samples at 4oC in 50 ml (sterile) centrifuge tubes or zip-top bags.
- 1 g of soil samples should be suspended in 10 ml of 0.85% NaCl.
- 10 minutes of shaking heating at 70°C.
- Aliquots of 100l of suspension should be spooned onto nutrient agar (1.5% agar, 0.5% peptone, 0.3% beef extract, and 0.5% sodium chloride).
- For 48 hours, incubate plates at 30°C.
- Subculture bacterial colonies displaying a Bt-like phenotype once more on brand-new plates, then incubate.
- Use Ziehl’s carbol fuchsin and amino black to stain the culture, then examine it under a regular light microscope to make a preliminary identification.
Expected Outcomes of Bacillus Thuringiensis Isolation
- Most colonies have an overall matte white color that is flat, dry, and has uneven borders.
- Cultures that exhibit parasporal crystals that are stained black under a microscope may be important and need to be stored.
Gram Staining
Gram Staining
Gram Staining: What is it?
Gram staining is a differential bacterial staining method used to distinguish between Gram Positive and Gram Negative types of bacteria based on the makeup of their cell walls.
It is both the most used and significant staining method in bacteriology, particularly in medical bacteriology. It is frequently the initial test carried out on bacteria during the process of their identification and observation.
Two stains are used in this staining method
Safranine as a counterstain and crystal violet as the primary stain. Gram-positive bacteria will retain their primary stain and appear violet or purple. Gram-Positive bacteria are what these organisms are known as.
Under a microscope, the Gram-Negative bacteria in the other group will lose their primary stain and take up the counterstain, making them appear pink or red. Gram-Negative bacteria are what these organisms are known as. The differential staining technique is named as such because it allows bacteria to be divided into two categories.
History of Gram Staining
Hans Christian Gram, a Danish bacteriologist, developed this method in 1884. (1853 September 13 to 1938 November 14). He created this staining method to find the pneumonia-causing bacteria. The division of bacteria into Gram Positive and Gram Negative kinds later gained popularity.
Gram Staining Theory
Based on the variations in the composition and structure of bacterial cell walls, gram staining and differentiation are performed. Bacteria with extensive peptidoglycan layers on their cell walls will resist the primary stain’s ability to fade, remaining violet or purple in hue. Bacteria with a thin peptidoglycan coating and little cross-linkage gain a pink or red counterstain in place of the primary stain following decolorization.
IMViC Test
IMViC Test
The IMViC test is a collection of four distinct biochemical assays used to detect and distinguish bacteria, particularly those belonging to the Enterobacteriaceae family. It may (and is) used to identify any sort of bacterium, however, it is primarily employed to identify Gram-negative bacteria. It is essential for distinguishing and classifying members of the Enterobacteriaceae family.
The abbreviation IMViC stands for four separate biochemical tests, with the exception of “I,” each letter standing for a different test in this series. The following biochemical tests are included in the IMViC series
“I” for Indole Test
“M” for Methyl Red (MR) Test
“Vi” for Voges-Proskauer (VP) Test
“C” for Citrate Utilization Test
We can examine six biochemical properties by making a slight alteration, such as using SIM (Sulfide-Indole-Motility) agar medium in place of 4. The ability to assess bacteria’s “motility” and “sulfide production” (H2S production) ability is revealed by the usage of SIM medium. As a result, it is a set of biochemical testing that is universally accepted.
The most popular primary biochemical test series is IMViC. Although crucial for distinguishing Enterobacterales, it is also utilized to characterize and identify a number of Gram-positive bacteria. It is frequently applied for both teaching and research objectives in clinical laboratories. Each test in the series is simple to administer and provides results in 24 to 48 hours. employed as a major screening method as result.
IMViC test objectives
- To characterize and identify isolated unknown bacteria by looking at some of their biochemical traits, such as indole generation, acid production, acetylmethylcarbinol (acetoin) production, and citrate consumption.
- To distinguish and recognize specific members of the Enterobacteriaceae family.
IMViC Test Principle
The IMViC test is based on variances in the metabolic needs and characteristics of various bacterial genera and species. The series’ “indole test” and “citrate utilization test” look for bacteria’s capacity to make and use particular enzymes and nutrients, respectively. The ‘MR test’ and ‘VP test’ in the series, on the other hand, identify the ultimate metabolic byproducts created by the bacteria using a given food. The test bacteria are cultivated in a specific culture medium for this purpose; several media are used for various tests (except MR and VP tests which need the same culture media; MR-VP media).
IMViC Test Requirements
The IMViC test is a collection of various biochemical assessments that call for various culture mediums and reagents. Broths were the only media utilized in the past, however diverse solid media are now highly recommended for their simplicity and versatility in testing various qualities.
Indole Test prerequisites
Culture media
Historically, tryptophan broth was employed.
Sulfide – Indole – Motility (SIM) medium has recently gained a lot of popularity (because it gives the result of H2S production and motility also).Also suggested is the Motility, Indole, and Urea (MIU) medium (because it gives the result of urease production and motility also).
Reagents
- The most commonly used is Kovac’s Indole Reagent, which is a 4-(dimethylamino) benzaldehyde and hydrochloric acid solution in n-butanol or amyl alcohol. Aerobic organisms prefer it.
- For anaerobic and weak indole-producing organisms, Ehrlich’s Reagent (a mixture of p-dimethylaminobenzaldehydre and hydrochloric acid in ethyl alcohol) is preferable.
- For the spot indole test, use 5% p-dimethylaminobenzaldehyde or 1% p-dimethylaminocinnamaldehyde in 10% (v/v) concentrated HCl.
Requirements for the MR test include
Culture Media MR-VP broth
Reagents Methyl red indicator
Requirements for the VP Test include
Culture Media MR-VP broth
Reagents VP Reagent I (5% – -Naphthol Solution) or Barritt’s A Solution
VP Reagent II (40% KOH solution) or Barritt’s B solution
Requirements for the Citrate Test include
Culture Media Simmon’s Citrate Agar
Reagents Simmon’s citrate medium already contains the bromothymol blue indicator.
Although it is developed, the streamlined IMViC agar plate with modified media that contains all four IMVic test media is not generally used.
Describe the Indole Test
The IMViC test series includes a biochemical test called the indole test that looks for bacteria’s capacity to create indole as a byproduct of tryptophan metabolism. The IMViC’s letter “I” serves as a visual cue.
Basics of the Indole Test
Tryptophan is an amino acid that some bacteria may generate the enzyme “tryptophanase” to assist them to break down into “indole, pyruvic acid, and ammonia.”
When added to a medium containing a bacterial culture that has produced indole, the indole reacts with the aldehyde in the indole reagent to produce a distinctive color.
A pink-to-red quinoidal compound occurs, giving rise to a pink-to-red color ring if the reagent contains benzaldehyde.
When cinnamaldehyde is present in the reagent, a green-to-blue color ring develops, which results in the creation of a blue-to-green molecule.
Benzaldehyde and amyl alcohol are both used in Kovac’s indole reagent. The top develops a cherry-red or pink-red ring due to the presence of amyl alcohol, which leaves an oily coating and is insoluble in water.
Tryptophan-containing culture media is used to cultivate test microorganisms for 24 to 48 hours, after which the result is read using an indole reagent. Depending on the reagent type utilized, the development of a pink-to-violet-red or green-to-blue color ring indicates a favorable outcome. A lack of color change or the emergence of a faintly yellowish color ring at the top are indicators of a negative color.
Indole Positive Bacteria
Escherichia coli, Klebsiella oxytoca, V. cholerae, Proteus vulgaris, Porphyromonas asaccharolytica, Vibrio spp., Flavobacterium spp., Providencia spp., Enterococcus faecalis, Haemophilus influenzae, Morganella morganii, Aeromonas spp., Citrobacter koseri
Indole Negative Bacteria
Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Shigella spp., Citrobacter freundii, Pseudomonas aeruginosa, Bacteroides fragilis, Staphylococcus aureus
Methyl Red (MR) Test: What Is It?
A biochemical test called the Methyl Red (MR) Test can be used to identify bacteria’s capacity to create stable mixed acids as metabolic byproducts of glucose metabolism. The IMViC’s letter “M” serves as a clue.
Basics of MR Testing
The mixed acid fermentation pathway is used by several bacterial species to metabolize glucose. They transform pyruvate into stable mixed acids by using this metabolic process.
When grown in a medium containing glucose (or another carbohydrate), these acid-fermenting bacteria will release acids, lowering the pH of the medium to 4.4 or below. A methyl red indicator added to a medium containing such acid fermenters will cause the medium to turn red.
The 24-hour incubation period is followed by the addition of a methyl red indicator to the MR-VP broth. A favorable outcome is indicated by the increase of red color, whilst an unfavorable outcome is indicated by the emergence of a yellowish hue.
MR Positive Bacteria
Escherichia coli, Salmonella spp., Shigella spp., Citrobacter spp., Proteus spp., Yersinia spp., Edwardsiella spp., Staphylococcus aureus
MR Negative Bacteria
Klebsiella pneumoniae, Enterobacter spp., Hafnia spp., Serratia marcescens
What is the VP Test (Voges-Proskauer)?
The IMViC test series includes the biochemical Voges-Proskauer (VP) Test, which identifies the presence of bacteria that can convert pyruvate into the neutral intermediate product acetylmethylcarbinol or acetoin. The IMViC’s letter “V” serves as a visual cue.
Fundamentals of VP Test
During the butanediol pathway of 2,3-butanediol synthesis, pyruvate can be converted into the neutral intermediate product acetyl methyl carbinol, also known as acetoin.
If acetoin is present in the medium, air and KOH will quickly oxidize it to diacetyl. In the presence of -naphthol, the guanidine component of peptone reacts with the resulting diacetyl to yield a pink to the red-colored product. VP reagents I and II are introduced after the 48-hour aerobic incubation on MR-VP broth, and the color change is visible within 30 minutes. The appearance of pink or red color at the top of the soup right away, within 30 minutes, but no later than an hour, is a sign of a successful outcome. A negative VP test is represented by no change in color.
VP Positive Bacteria
Klebsiella spp., Enterobacter spp., Viridans Streptococci (except S. mitis, and S. vestibularis), Proteus mirabilis, Hafnia spp., Serratia spp., Staphylococcus aureus
VP Negative Bacteria
Escherichia spp., Proteus vulgaris, Citrobacter freundii
Citrate Utilization Test: What is it?
The IMViC test series includes a biochemical test called the Citrate Utilization Test that measures an organism’s capacity to use citrate as its only source of energy. The IMViC’s letter “C” serves as a clue.
Citrate Utilization Test Principle
Citrate can be the only source of carbon for some bacteria. Citrate is broken down into acetic acid and oxaloacetic acid by the citrase enzymes produced by these bacteria. Following the decarboxylation of the oxaloacetic acid, pyruvate and CO2 will be produced.
Alkaline “sodium carbonate” will be created when released CO2 combines with water and extra sodium from sodium citrate. The pH of the medium will rise thanks to the sodium carbonate.
CO2 + H2O + more sodium from sodium citrate → Na2CO3 (alkaline)
Furthermore, CO2 emissions will start the metabolism of ammonium salts. Ammonia will be produced if the ammonium salts are used as a source of nitrogen (or ammonium hydroxide).
Ammonium salt → Ammonium hydroxide (alkaline)
Ammonium hydroxide and sodium carbonate working together will raise the media’s pH above 7.6. The pH indicator bromothymol blue in the media will change from deep forest green (at a neutral pH) to Prussian blue as a result of the increase in pH.
After 24 to 48 hours of incubation (up to 4 days for some), bacterial growth and color change are seen in the slanted part. Growth and a switch from green to a deep blue hue of the slant are signs of a successful outcome. If the color of the slant does not change, the outcome is negative.
Citrate Positive Bacteria
Klebsiella spp., Citrobacter spp., Serratia marcescens, Proteus mirabilis, Enterobacter spp., Salmonella spp. (except Salmonella Typhi and Paratyphi A), Edwardsiella spp., Providencia spp.
Citrate Variable Bacteria
Proteus vulgaris, V. cholera, V. parahaemolyticus.
Citrate Negative Bacteria
Escherichia coli, Shigella spp., Salmonella Typhi and Paratyphi A, Yersinia spp. Morganella morganii, Staphylococcus aureus, etc.
The IMViC Test’s Uses
- It is frequently used to describe and identify unidentified isolated bacteria up to the genus level in clinical, research, and teaching laboratories.
- The test can distinguish between and identify the members of the Enterobacteriaceae family, in addition to the Urease test and the TSI (Triple Sugar Iron) test.
- To distinguish between species within a genus, some tests are performed. Examples include distinguishing between K. oxytoca and K. pneumoniae, C. koseri and C. freundii, P. vulgaris and P. mirabilis, etc. using the indole test.
Foodborne Infection by Shigella – Shigellosis Food Poisoning
Foodborne Infection by Shigella - Shigellosis Food Poisoning
Describe the Shigella infection
The ideal and the colonic epithelium is harmed by acute gastrointestinal tract infections caused by Shigella species, which also result in bacillary dysentery. Developing and undeveloped nations with low sanitation, hygiene, and medical facilities are where shigella infections are most frequently recorded.
- Shigellosis has a significant death rate, particularly in malnourished children under the age of five.
- Due to the significant loss of fluids and blood during diarrhea, which might cause mortality, the Shigella infection sufferer experiences decreased nutrition.
- S. dysenteriae (serogroup A), S. flexneri (serogroup B), S. boydii (serogroup C), and S. sonnei are the four serogroups that Shigella species are divided into (serogroup D).
- Among them, S. dysenteriae Type 1 is in charge of deadly epidemic outbreaks, whilst other serotypes are in charge of little and uncommon illnesses.
Biological characteristics of Shigella
- Gram-negative
- Facultative anaerobes
- Non-sporulating
- Non-motile
- Non-encapsulated
- Prokaryotic rods
- Optimum temperature – 7 to 46°C
- Can resist 5% NaCl and pH
- Can survive harsh physical and chemical conditions
- Sensitive to pasteurization temperature
Shigella infection sources and routes of transmission
- The human host’s digestive system serves as the primary reservoir for Shigella species.
- Rarely does Shigella infection occur in animals.
- The fecal-oral pathway or person-to-person contact is the route of transmission.
- The main sources include drinking contaminated water and eating tainted food.
- Fruits and vegetables are contaminated by the contaminated irrigation water used by farmers.
- Due to a lack of sanitary facilities and adequate hygiene practices, children and newborns are more susceptible to shigellosis, particularly in impoverished and developing nations.
Infection with Shigella Epidemiology
- The Japanese microbiologist Kiyoshi Shiga came up with the name Shigella dysenteriae while looking into a severe dysentery epidemic that struck Japan in 1896.
- Shigellosis can be brought on by just 10 to 100 bacterial cells, as opposed to the 1000–100,000 required by other diseases.
- The World Health Organization (WHO) estimates that there are 165 cases of shigellosis worldwide each year, with 55,000 child deaths under the age of five.
- A sporadic outbreak of S. dysenteriae and S. flexneri, which were isolated from an infected person’s stool sample, occurred in Kolkata.
- Epidemics and endemic diseases have primarily affected South Asia and East Africa in recent decades.
Shigella-related illnesses and signs and symptoms
- The onset of symptoms is typically 1 to 3 days after ingesting bacterial cells, while it can be anywhere between 12 hours and 7 days depending on the dose.
- Fever, appetite loss, abdominal pain, bloody or watery diarrhea, colon inflammation, weariness, malaise, and fever are typical symptoms.
- Dehydration may happen as a result of the outflow of too much watery fluid, but it rarely does and shouldn’t be a major issue.
- Malnutrition and anorexia, which are the leading causes of death in children under the age of five, require aggressive management.
- Some patients may experience neurological issues such as headaches, drowsiness, and abnormal body movement.
- The illness goes away on its own in 5 to 7 days, but an infected individual may continue to pass bacteria in their feces while asymptomatic and provide a risk of infection spreading.
Shigella infection toxicity mechanism
- When the organism enters the body through the mouth and travels to the large intestine, the infection begins.
- Due to the increased mobility of epithelial cells and rapid liquid flux, Shigella can withstand the stomach pH and does not cling to the small intestine.
- Once inside the intestinal epithelial layer, pathogens are bound by M cells in vacuoles.
- After escaping, the infection moves on to the macrophages, where it is phagocytized and causes apoptosis.
- Shigella reproduces quickly in the large intestine and spreads to neighboring cells as it travels through the epithelial layer, inflicting inflammation and tissue damage.
- Due to the secretion of the proinflammatory cytokine IL-12, which causes necrosis in the host body, S. flexneri is known to kill mitochondria.
- The pathogens are encased by a plasma membrane and begin intracellular reproduction and cell-to-cell bacterial dissemination during inter and intracellular (cell-to-cell) migration.
- According to reports, the immune system of the host is what triggers the inflammation and ulceration of the mucosal layer; the pathogen’s intracellular proliferation is not a factor.
- Shiga toxin is a powerful toxin that produces bloody diarrhea and is known to be produced by S. dysenteriae.
- The host cells die as a result of the toxin’s termination of the protein production pathway after entering the host cell via endocytosis.
- The gram-negative bacteria’s lipopolysaccharide (LPS) layer also functions as an endotoxin that only becomes active when the cell is destroyed and harms the epithelial tissues.
Shigella laboratory diagnostic
Methods for bacterial cultivation
- Dysentery patients should have a stool sample collected and examined right away because the organism only survives outside the host body for a brief period of time.
- To isolate the bacteria, the sample to be examined should be taken during the very beginning of the infection.
- Xylose-Lysine Deoxycholate Agar, MacConkey Agar, and Salmonella-Shigella Agar are the specific media used to culture Shigella.
Bioassays
- To measure the impact of the pathogens, various animals including guinea pigs, rats, rabbits, and monkeys are given oral medications.
- Some had obvious eye inflammation that progressed to massive intestinal tissue damage.
- Animal use is nevertheless restricted because of moral concerns.
Immune system tests
- There are kits for enzyme immunoassay (EIA), also known as a dot blot assay, which is used to detect infections.
- EIA kits typically cost little money to use and have a 94% efficiency rate.
- Dipstick immunoassays, the Wellcolex Color Shigella test, and the latex agglutination test are further immunoassays (WCT-Shigella).
Using molecular methods
- The most reliable method to quickly find the Shigella gene is matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).
- Conventional PCR improved the diagnosis by 50% by identifying the different Shigella species.
- Target genes like ipaH, virA, iral, LPS, and plasmid DNA can be found by them.
Taking care of shigellosis
- Shigella can become resistant to antibiotics, which might make it difficult to treat a patient with shigellosis symptoms.
- In a clinical trial, ciprofloxacin, ampicillin, sulfamethoxazole, and nalidixic acid were used to demonstrate their efficiency against Shigella.
- Azithromycin and fluoroquinolones are the antibiotics of choice for shigellosis at the moment.
- Treatment should begin early in the course of the sickness and is dependent on how severe the infection is.
- For an infected person to replenish the electrolyte loss, oral rehydration and intravenous fluid therapy are frequently used as treatments.
Shigellosis Prevention and Control
- Shigellosis can be prevented most successfully by using proper sanitation, decent personal hygiene, and proper feces disposal.
- Given that shigellosis is spread by the fecal-oral channel, it is advisable to prevent someone from preparing food if they are displaying signs of a digestive issue.
- Utilizing chlorinated water to wash produce before eating.
- Food goods should be refrigerated properly to prevent bacterial growth.
- Shigellosis typically occurs in undeveloped and underdeveloped nations, so it is important to educate people about proper hygiene and sanitation, to avoid consuming feces-contaminated food and water, and to consume nutrient-rich meals while ill in order to reduce the number of cases.