Citrate Utilization Test

Objective:
-To determine the ability of bacteria utilize citrate as sole carbon source.
T-o use this fact to differentiate enteric organisms.

Principle:

           

            Some organisms are utilizing citrate as a carbon source for their energy.  The enzyme citrate permease facilitates the transport of citrate into the cell.  Then citrate is acted upon by enzyme citrase which produces oxaloacetic acid and acetate. That are further converted to pyruvic acid and CO_2. This CO₂ combines with medium (Simmon’s Citrate) and water to form sodium carbonate (NaHCO₃) and ammonia (NH₃). This products change the pH of the medium into alkaline. Bromothymol blue (indicator) turns to deep prussian blue in alkaine pH. At neutral pH , bromothymol is green.

Materials required:
 Bacterial cultures (Klebsiella sp., E.coli)

 Simmon’s Citarte Agar

Procedure :
1.Inoculate the bacterial cultures on simmon’s citare agar tubes (slant) by stab and streaking.
2.The tubes are incubating at 37° C for 24 hrs.
3. To observe and record the result based on color change.

Observation:
-  Prussian blue /deep blue color change in  Klebsiella sp tube 
- Green color / No color change in tube with E.coli

Result : -
 - Klebsiella sp  : Citrate  Positive 
- E. coli : Citrate Negative

 Interpretation:

           

            Klebsiella sp., using citrate as sole carbon source with help of citrate permease and releases sodium carbonate and ammonia and this products change the pH of the medium into alkaline. In this pH condition the indicator bromothymol blue change the color from green to deep blue/Prussian blue and give positive result.  E.coli doesn’t produce the enzymes like citrate permease and citrase, which indicate the negative result and no change in medium color. 

 Citrate Positive Organisms:

Klebsiella sp., Enterobacter sp., Proteus mirabilis, 

                                                                   Citrate Positive

                                                                        Citrate Negative

Composition of Simmon's Citaret agar :

Ingredients Per liter of distilled water

Sodium Citrate (dehydrate)    -   2.0 g

Ammonium dihydrogen
 phosphate -   1.0 g

Dipotassium phosphate -   1.0 g 

Magnesium sulfate  (heptahydrate) -   0.2 g

Sodium chloride (NaCl) -   5.0 g

Bromothymol Blue (indicator) -   0.08 g

Agar  -   15.0 g

pH : 6.9

        
  

                     

Summer Research  Fellowship Programme-  2018

Science Academie's summer research fellowship  progrmme 2018  for Students and teachers.

Internship in various well reputed  central government laboratories  and universities in India with stipend 

May undergo short - term projects there with exposure.

Please go through the following website...


http://web-japps.ias.ac.in:8080/fellowship2018/application_instructions.jsp



last date : 30 November 2017.. through online...

Khorana Scholarship Programme
The Department of Biotechnology (DBT), Govt. of India,  Indo-US Science and Technology Forum (IUSSTF) and WINStep Forward are partnering to support the prestigious Khorana Program for Scholars. The Khorana Program will provide opportunities to Indian students to undertake research at University of Wisconsin-Madison (UW) and partner universities in Summer 2018 for a period of 10 weeks. A list of UW partner universities is available http://www.winstepforward.org The Khorana Program is envisaged to Provide encouragement to young scholars to undertake R&D Enable students to carry out research at a premier University in the United States Transform research into societal benefits Build a seamless scientific community between India and the United States Eligibility: B.Tech, M.Tech and B.Sc./M.Sc. students currently enrolled in recognized institutions of higher education in India in Biotechnology and allied areas are eligible to apply. Students pursuing Ph.D/Final year students are not eligible to apply. Scholarship includes: Stipend, Airfare, Health Insurance Program dates: May – July 2018 For immediate answers to your questions you may contact: Dr. Nishritha Bopana Indo-US Science and Technology Forum (IUSSTF) 12 Hailey Road, Fulbright House, New Delhi- 110 001 Phone: 91-11- 42691713, 42691700, 42691708 Fax: 91-11-23321552 E-mail: scholar@indousstf.org  Application deadline: 30 November 2017 Further details go to the following link /;  http://www.iusstf.org/story/53-50-Khorana-Program.html

SSOP : food Microbiology Quality control

1.What is an SSOP? 
An SSOP is a written procedure that explains exactly how a certain cleaning task is completed. These procedures may vary from farm to farm. The purpose of the SSOP is to provide enough detail so employees can perform the task correctly by reading the procedure without any additional instruction. It also shows an auditor exactly how the task is performed.

2.
Completing an SSOP : To complete an SSOP, you need to write down step‐by‐step instructions, which include frequency, monitoring, record keeping, corrective actions and confirmation signature. The key elements that must be included in the step‐by‐step instructions are:
1. Equipment used (e.g., hoses, cloths, buckets, pressure washer, scrub bushes, brooms).
2. Chemicals used (if any) as well as concentrations and mixing instructions (e.g., sanitizers, soaps, is it mixed with water, sprayed on, how much chemical, how it is measured).
 3. Step‐by‐step procedures clear enough that you could do the cleaning without having to ask any questions.

Procedure details should include the following: General Information
 • Identify and explain the use of soap or sanitizers and their concentrations and equipment used. Please consult the “Resources” page for Best Practice information on use of chlorine for Fluming and Cleaning Fresh Fruits and Vegetables and Cleaning Packinghouse Equipment.

• Must explain that equipment must not be used for livestock/poultry slaughter or meat processing activities if there is a potential for this to occur at your facility. Frequency

• Before each use of equipment, a general inspection is conducted to ensure equipment does not contribute to the contamination of product (e.g., checks for leaks, broken, loose, corroded or damaged parts, chipping paint, rust, rotting wood, cleanliness)

• Weekly (at a minimum when in use) – Producer/storage intermediary/packer inspects equipment in direct contact with produce (e.g., grading table, packing line, buncher, baggers), or that may have an impact on food safety for proper functioning (e.g., checks for faulty or loose parts). Weekly (at a minimum when in use) – Producer/storage intermediary/packer ensures that building equipment in direct contact with product is clean by at least one of the following cleaning procedures:

1. Water with friction (e.g., pressure wash, wiping, and/or scrubbing)
 2. Water and sanitizer (e.g., chlorine and/or quaternary ammonium)
 3. Water and soap
 4. Dry cleaning (e.g., broom, brushes, and/or air)

 Record Keeping

 • The inspection should be recorded.
• For an example Equipment Cleaning, Maintenance and Calibration form, please consult the “Forms and Training Materials” page. .


Examples :

Example A: 1. Use the black hose in the packing barn to rinse the grading table.
2. Use the purple scrub brush to scrub the surface of the table.
3. Use the hose again to rinse the grading table a second time.
4. Allow the table to dry before use.
5. This procedure should be performed every time before use of the grading table.
6. Record that you performed this activity on the Equipment Cleaning, Maintenance and Calibration form.

Report the date and time performed on the sheet and sign it once completed. Example B (using cleaning chemicals):
 1. Connect the pressure washer to the tap in the packing barn closest to the grading line.
 2. Use the pressure washer to wash down the entire grading line. Start at the top of the machine and work your way down.
3. Ensure that all visible organic matter, including leaves, dirt and other debris are removed from all parts of the grading line.
4. Fill the bucket with water from any tap in the packing barn.
 5. Add two teaspoons of bleach to the bucket.
 6. Use the bleach solution and a clean cloth to wash down the stainless steel grading tables. Ensure that the solution is in contact with the table for at least two minutes.
7. Allow to air dry.
8. This procedure should be performed every time before use of the grading line.
 9. Record that you performed this activity on the Equipment Cleaning, Maintenance and Calibration form. Report the date and time performed on the sheet and sign it once completed.

Food Microbiology : HACCP Principles

Click the link below....


https://docs.google.com/presentation/d/1-BJPK4OKSi2SYSWk5CbYOlAiNHWKFh50nxApk18Zvgg/edit?usp=sharing

Food Microbiology: GMP, SSOP PPT

Please click following link to see the ppt.

https://docs.google.com/presentation/d/1JsyGwBMq4UhNminNjzZZmY-KJsrS1mcI8FpR51DNk9o/edit?usp=sharing

Voges- Proskauer (VP) Test (Non – acid end product test)

Objective:

    To determine the ability of microorganism produce non- acidic or neutral end products from glucose.

Principal:

            Some bacteria digest glucose to form key intermediate pyruvic acid which can further be metabolized to produce acetoin (acetyl methyl carbinol) as an intermediate and this acetoin further reduced to form 2,3-butanediol. The detection of 2,3, butanediol is difficult, so the test detects the intermediate acetoin by addition of 40 % potassium hydroxide (KOH) and 5 % alpha napthol solution (Barritt’s reagent) . The presence of acetoin and reagent , develops crimson red color.

           

Materials Required:
            -Bacterial Culture (Klebsiella sp., and E. coli)

            -MR-VP broth

           -Barritt’s Reagent : (40 % potassium hydroxide (KOH) , 5 % alpha napthol solution and 0.5% creatinine)

Procedure:
1.      Inoculate loopful of bacterial culture into MR-VP test tubes 
2.      Test tubes are incubating at 37° C for 24 hrs.     
3.   The next day add 0.6 ml of alpha napthol (Barritt’s reagent A)to the tubes and mix thoroughly. 
4.      Add 0.2 ml of 40 % KOH (Barritt’s reagent B) to the culture tube and shake vigorously.
5.      After 15 min. examine the color formation in the culture tubes.

Observation:  
- Pink color formation and turns crimson red color formation within few minutes in Klebsiella sp., tube.
 -Yellow or copper color formation in E.coli tube.

Result: 
-Klebsiella sp.,  is VP positive (present of acetoin)
-E. coli is VP negative ( acetoin absent)

Interpretation:

          If the culture produces acetoin, it is spontaneously oxidized to diacetyl in the presence of oxygen and potassium hydroxide (alpha napthol is used to increase the sensitivity of the reaction). Diacetyl then reacts with creatinine to form a pink colored complex and within few minutes it forms crimson red color.

VP Positive Organisms :

         Klebsiella sp., Enterobacter

MR/VP medium Composition:

Peptone    5g

Dipotssium hydrogen phosphate    5g

Glucose (10% solution)         50 ml

Distilled water 1000ml           

Barritt’s Reagent:

Solution A:    40 % potassium + 0.5 g creatine

Solution B:   5 % solution of α-napthol in absolute alcohol 

METHYL RED (MR) TEST (Mixed Acid Fermentation)

          

Objective:
 -  To determine whether the organisms performs mixed acid fermentation by using glucose.
 - To identify the bacteria producing stable acids by fermentation.
 -  To differentiate between all glucose oxidizing enteric organisms.

Principle:

            The glucose is the major substrate oxidized by all enteric organisms for energy production. The end products of this process vary depending on the enzymatic pathways present in the bacteria. The pH indicator methyl red detects acid range as a result of acid end products. This test is value to differentiate Escherichia coli and Enterobacter aerogenes.  E.coli (Mixed acid fermenters) produces a mixture of fermentation acids such as lactic, acetic, succinic and formic acids and thus acidifies the medium. E. aerogenes (Bautanediol fermenters) forms neutral end products like butanediol, acetoin so the pH is elevated to approximately 6.

            The pH is indicates by methyl red indicator, it turns red color in the pH range of 4. At  a pH of 6 still acidic but a lower hydrogen ion concentration. The indicator turns yellow which is a negative test.   

Materials required:

Cultures : E.coli, Klebsiella sp., and E. aerogenes

Medium :  MR-VP broth

Indicator : 0.02% methyl red reagent

Procedure : 
  -   Inoculate one loopful of test culture into the test tubes of MRVP broth.
 - The tubes are incubating at 37° C for 24 hrs.
  - After Incubation add 5-6 drops of methyl red reagent to the culture tubes and thoroughly mixed.
 - To determine and record the result based on color change.

Observation:
-Red color formation in  tube with E.Coli
-Yellow color in Klebsiella sp., and E. aerogenes tubes.

Result :
 - E.coli  : MR  Positive 
  - E. aerogenes  and Klebsiella sp.., Gives Negative Result in Methyl Red test. 

Interpretation:

            When microbes ferments glucose releasing acids, the hydrogen ion concentration increases i.e., the pH goes down.  The pH indicator methyl red is yellow above pH 6.3 and red below 4.2 pH.  E. coli  Shows a red color indicating acidic nature of the medium due to acidic end products.   The organisms like Klebsiella sp., and E. aerogenes are not producing acid end products so lack of color change and maintains yellow color.

MR Positive Organisms:

E.coli, Salmonells Sp.,

MR Negative Organisms:

Klebsiella Sp., E. aerogenes and Pseudomonas sp., 

MR/VP medium Composition:

Peptone    5g

Dipotssium hydrogen phosphate    5g

Glucose (10% solution)         50 ml

Distilled water 1000ml           

Methyl Red : 
Methyl red    0.1g
ethanol   300 ml
distilled water 200ml

Indole Test

Objective:
-To determine the organism is producing indole from amino acid tryptophan
-To realize that amino acid can be used by microorganisms as energy source
-To  identify the tryptophanase enzyme producing organism

Principle:

            Tryptophan is an amino acid that can undergo deamination and hydrolysis by tryptophanase enzyme which is produced by bacteria.  Indole  is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid.  Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.

        When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-dimethylaminobenzaldehyde(p-DMAB) in amyl alcohol) the solution turns from yellow to cherry red. Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the top of the broth.  

Materials required:

Cultures : E.coli, Klebsiella sp.,

Medium :  Tryptone/Peptone broth

Indicator : Kovac’s reagent

Procedure :

1   -Inoculate one loopful of test culture into the peptone broth tube.

2   -The tubes are incubating at 37 ͦ C for 24 hrs.

3   -The next day gently add Kovac’s reagent into the culture (ratio 1:10, that means 0.5    ml into 5 ml of cultue)

4   - Mix well and wait for upto 5 minutes to develop the color.

Observation:
-  Cherry red color formation on top layer of the E.Coli broth.

-  No color change (Yellow color) in Klebsiella sp., broth.

Result :

-  E.coli  : Indole Positive 

- Klebsiella sp., :   Indole Negative 

Interpretation:

          E.coli utilizes tryptophan with the help of tryptophanse enzyme and produces indole positive,it gives cherry red color when react with Kovac’s reagent . The absence of a red color indicates tryptophan was not hydrolyzed by bacteria (Klebsiella sp.,) are indole negative.

Indole Positive Organisms:

E.coli., Proteus vulgaris, Enterococcus faecalis

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Kovac’s Reagent:

Amyl alcohol or isoamyl acohol         150 ml

Paradimethyl aminobenzaldehyde     10g

Conc. Hcl     50 ml

Photosynthesis