Anthrax panic, 2001

First anthrax-laced letter is mailed on 18 Sept, 2001
Florida sees first of five deaths, three weeks later
Panicked Americans try to stock up on antibiotic Cipro
Postal depots shut for de-contamination
Senate offices shut for weeks
Hoaxes become an almost daily occurrence
Plans to deal with a biological weapons attack updated
Mail irradiated to kill anthrax spores


August 1, 2008 BBC News
US anthrax ‘suspect’ found dead
A top US scientist suspected of anthrax attacks in 2001 has apparently killed himself just as he was about to be charged
The Los Angeles Times said Bruce Ivins, 62, had taken an overdose of painkillers (a massive dose of a prescription Tylenol mixed with codeine). It said he had recently been told of the impending prosecution.
There has been no official comment but unnamed sources said prosecutors were to indict and seek the death penalty.
Five people died when anthrax was posted to the media and politicians.
The incidents took place shortly after the 11 September attacks in 2001.
Security measures in the wake of the anthrax attacks increased the public’s fear of their vulnerability to terrorism.
As well as the five deaths, 17 other people were made ill.

Progress
Dr. Ivins worked for the US Army Medical Research Institute of Infectious Diseases (USAMRIID) in Fort Detrick, Maryland.
In 2003, Dr Ivins was awarded the highest honor for defence department civilian staff for his anthrax vaccine work.
The authorities were investigating whether Dr. Ivins has released the anthrax to test the vaccine he was developing.
Dr. Ivin’s lawyer, Paul Kemp, said: “We assert his innocence in these killings, and would have established that at trail.”

Depression
The FBI focused more on Dr. Ivins after the leadership of the investigation was changed in 2006.
The investigation had been making new progress based on analysis of anthrax-tainted letters posted to senators.
There has been no official comment but unnamed sources said prosecutors were to indict him and seek the death penalty.



Monday, August 4, 2008 Cox News Service
Army microbiologist who committed suicide was to be indicted in anthrax cases
An award winning army anthrax expert killed himself this week, as federal prosecutors prepared to indict him on charges stemming from the lethal anthrax attacks that shook the country seven years ago, officials said Friday.
The scientist Bruce Ivins, helped develop a new generation of anthrax vaccine in his 35 years as a microbiologist for the Army’s bio warfare lab outside Washington, said lab spokeswoman Caree Vander Linden.
His apparent suicide came five days after a Frederick Country issued a temporary restraining order against Ivins, after a social worker who was counseling him complained in court documents that Ivins had stalked, harassed and threatened to kill her.
In her petition for the restraining order, Jean Duley wrote that she had received a subpoena to testify Friday before a federal grand jury assessing evidence against Ivins. In handwritten testimony, Duley said Ivins “has a history dating to his graduate days of homicidal threats.”
Weeks after the September 11, 2001, terrorist attacks, letters laced with anthrax were mailed from a postal box in Princeton, N.J., to newsrooms in Florida and New York, as well as the Capitol Hill offices of two U.S. senators. The attacks panicked much of the nation and triggered one of the largest investigations in recent history.
Code named “Amerithrax,” the investigation has involved 17 FBI special agents and 10 U.S. Postal Service Inspectors. Together they’ve conducted 75 searches and more than 9,100 interviews in what the Justice Department called “relentless pursuit” of the perpetrators.
Ivins had special expertise in the production, purification, storage and use of anthrax spores.
Five years ago, he was one of three USAMIID scientists to receive the Decoration for Exceptional Civilian Service, the highest honor the Pentagon bestows upon civilian staff, for helping to resolve anthrax vaccine production problems.
“Awards are nice”, Ivins said at the time, “but the real satisfaction is knowing the vaccine is back on line.”
Ivins also reportedly helped the FBI analyze recovered from one of the tainted envelopes sent to a U.S. senator’s office in Washington.
However, he failed to report anthrax contaminations to Army officials for five months. In sworn oral and written statements to an Army investigator, Ivins said that he had erred by keeping the episodes secret from December 2001 to late April 2002. he said he had swabbed and bleached more than 20 areas that he suspected were contaminated by a sloppy lab technician.

Approaches

Remove misconception about making solutions

A solution that is 20% sugar is made sweeter by doubling the amount of sugar. What is the percent of sugar in new solution?
___________________________________________________
Be careful: wrong approach followed by most
Original solution: 20 gm sugar in 100 ml d/w
New solution: 40 gm sugar in 100 ml d/w
Percent of sugar in new solution: 40%
incorrect never do like this
____________________________________________________

Correct approach
Original solution: 20 gm sugar in 80 ml d/w
New solution: 40 gm sugar in 80 ml d/w
Percent of sugar in new solution:
33.33%

Now, let’s try another
Prepare 1% w/v sodium chloride (anhydrous) solution
1 gm sodium chloride (anhydrous) dissolved in small amount of water and final volume made 100 ml by adding d/w in volumetric flask of 100 ml (Read lower meniscus. Upper meniscus is read for colored solution).

Prepare 1% w/v sodium acetate (anhydrous) solution
1 gm sodium acetate (anhydrous) dissolved in small amount of water and final volume made 100 ml by adding d/w in volumetric flask of 100 ml.

What about preparing 1% w/v sodium acetate tri hydrate
It contains additional 3 molecules of water
Mol. Wt. of sodium acetate: 82
Mol. Wt. of 3 H20: 54
Total molecular wt of sodium acetate tri hydrate is 136
Therefore, 1.65 gm sodium acetate tri hydrate dissolved in small amount of water and final volume made 100 ml in volumetric flask by adding d/w.

Prepare 70% v/v ethyl alcohol from absolute ethyl alcohol
70 ml absolute ethyl alcohol taken in volumetric flask and d/w added to make final volume 100 ml. (about 30 ml d/w added to make final volume 100 ml).

Preparation of 1,000 ml of 10,000 PPM solution of sodium hypochlorite (5% available chlorine)
V1S1 = V2S2
V1 = 1,000 * 10,000 / 50,000 = 200
Therefore, 200 ml sodium hypochlorite mixed with d/w to make final volume 1000 ml in volumetric flask.


Molar solutions
Sodium chloride:
Mol. Wt. = 58.4
58.4 gm dissolved in d/w to make final volume 1 liter.
i.e., 58.4 gm in 1,000 ml = 1 mol/lt
5.84 gm in 100 ml = 1 mol/lt
0.584 gm in 100 ml = 0.1 ml/lt

Potassium hydroxide:
Mol. Wt. = 56
56 gm dissolved in d/w to make final volume 1 liter.
i.e., 56 gm in 1,000 ml = 1 mol/lt
5.6 gm in 100 ml = 1 mol/lt
0.56 gm in 100 ml = 0.1 mol/lt

Hydrochloric acid concentrated
Density = 1.19
Mol. Wt. = 36.5
Assay = 36%
D=M/V
V= M/D = 36.5/1.19 ml required for 100%
36.5*100/1.19 ml required for 1%
36.5*100/1.19*36 ml required for 36%
= 85.2 ml
85.2 ml HCl mixed with d/w to make final volume 1 liter
i.e.,85.2 ml in 1,000 ml d/w = 1 mol/lt
8.52 ml in 100 ml d/w = 1 mol/lt
0.852 ml in 100 ml d/w = 0.1 mol/lt

Sulphuric acid
Density = 1.84
Assay = 95%
Mol. Wt. = 98
V=M/D = 98/1.84 ml required for 100%
98*100/1.84*95 ml required for 95%
= 56.06 ml
56.06 ml H2SO4 mixed with d/w to make final volume 1 liter
i.e.,56.06 ml in 1,000 ml d/w = 1 mol/lt
5.606 ml in 100 ml d/w = 1 mol/lt
0.5606 ml in 100 ml d/w = 0.1 mol/lt
2.803 ml in 500 ml d/w = 0.1 mol/lt

NaOH
Assay = 97%
Mol. Wt. = 40 gm
40 gm required for 100%
40*100/97 required for 97%
41.23 gm NaOH mixed in d/w to make final volume 1 liter
i.e., 41.23 gm in 1,000 ml d/w = 1 mol/lt
4.123 gm in 100 ml d/w = 1 mol/lt
12.369 gm in 300 ml d/w = 1 mol/lt
1.2369 gm in 300 ml d/w = 0.1 mol/lt

NaH2PO4
Mol. Wt. = 156
156 gm mixed in d/w to make final volume 1 liter
i.e., 156 gm in 1,000 ml d/w = 1 mol/lt
1.56 gm in 100 ml d/w = 0.1 mol/lt
3.12 gm in 100 ml d/w = 0.2 mol/lt

Acetic acid
Specific gravity = 1.047
Mol.wt. = 60.05
V=M/D = 60.05/1.047 ml in d/w to make final volume 1 liter
= 57.35 ml
i.e., 57.35 ml in 1,000 ml d/w = 1 mol/lt
5.735 ml in 100 ml d/w = 1 mol/lt
0.5735 ml in 100 ml d/w = 0.1 mol/lt
1.147 ml in 100 ml d/w = 0.2 mol/lt

1.147 ml acetic acid mixed with d/w to make final volume 100 ml in volumetric flask


I hope it will be helpful for all those lost in confusion. Let me know whether it helped you or not. Thank you

E-test

E-test also known as epsilometer test is an exponential gradient testing methodology. The E-test which is quantitative method for an antibiotic sensitivity testing applies both dilution of antibiotic and diffusion of antibiotic into the media. E-test strip has concentration gradient of antibiotic with lowest concentration at lower region and highest concentration at topmost region. Following incubation, elliptical or symmetrical inhibition zone is formed. The intersection of the inhibitory zone edge and calibrated strip indicates the MIC value. It is simple, reliable and accurate method to determine MIC for wide spectrum of infectious agents.

Determining MIC by E test

Procedures for seroty

Procedures for Serotyping of Streptococcus pneumoniae by co-agglutination assay (modified) - Dr Rewa Kanungo (05.11.04)

Reagents:
1. 7.2 Phostpahte buffered saline (PBS)- 1000 ml with 0.05% Na Azide (for washing)
2. 1.5% formaldehyde- 10 ml
3. water bath 800C
4. 10 ml pipettes
5. centrifuge tube- x20
6. smaller glass tubes- x20
7. biochemical tubes –x20
8. micropipette- x20 (100 μl)
9. PBS (Ph 7.2)- 200 ml with 0.1% Na Azide
10. PBS (Ph 7.2)- 100 ml without Na Azide


Preparation of Staphylococcus aureus
1. In the morning, inoculate freshly cultured Cowan type 1 strain of Staphylococcus aureus into MHBA (6 plates)
2. Incubate at 370C overnight in CO2 jar.
3. Next day, wash plates with PBS (Ph 7.2) with 0.05% Na Azide)
4. Scrape gently with sterile glass rod.
5. Collect into a centrifuge tube
6. Centrifuge at 3000 x g for 20 min then discard supernatant
7. Combine pellets from all 30 tubes in 1 or 2 tubes
8. Depending on the volume of the pellet, add 2 times the quality of PBS (Ph 7.2) with 0.05% Na Azide to tube containing pellet.
9. Mix thoroughly
10. Centrifuge at 3000 g for 15 min then discard the supernatant
11. Add 10 ml fresh PBS (Ph 7.2) with 0.05% Na Azide to the tube containing the pellet
12. Repeat steps 10 & 11 three times
13. Add 1.5% formaldehyde 10 times the volume of the pellet to stabilize staph (e.g. to 1 ml pellet add 10 ml 1.5% formaldehyde)
14. Mix thoroughly and stand at room temperature for 90 min
15. Centrifuge at 3000 g for 15 min then discard the supernatant
16. Wash 3 times as before then discard supernatant
17. Add 10 ml of PBS (Ph 7.2) (wash buffer)
18. Put tube in 800C water bath for 5 minutes (to kill staph)
19. Wash 2 times
20. Make 10% suspension of pellet (to 1 ml pellet add 10 ml PBS Ph7.2 with 0.1% Na Azide
21. Transfer 1 ml from pellet suspension to 2 tubes , keep 1 aside as control
22. 1st tube: Make 2.5% suspension as control (not sensitized) and keep aside
23. 2nd tube to be used for sensitization


Sensitization Steps:
1. Add 0.1 ml of antiserum to a fresh, dry, sterile tube
2. Add 1 ml of 10 % pellet suspension (2nd tube) to the antiserum (drop wise) while shaking the tube.
3. Stand at room temperature for 30 min with intermittent shaking
4. Centrifuge at 3000 g for 15 minutes then discard supernatant
5. Add 5 ml PBS (Ph7.2) with 0.1% Na Azide (this will make a 2% solution) and distribute in 1 ml
6. Label the coagglutination antisera solutions appropriately (A-F,H, and P-T)
7. Store at 2-80C (i.e. refrigerate)


Test Procedure
1. Carry out a clean glass slide
2. Make 25 μl of the culture suspension in PBS (Ph 7.2) ( without Na Azide)
3. Add each of the pool coagglutination antisera solutions (25 μl) as per the chart provided with the Pneumostat kit ( A-F, H, and P-T)
4. Rock the slide by hand for 2 minutes
5. Observe the results
6. Test is positive if cell clumping is observed along with clearing of suspension within 2 minutes and control slide shows no clumping

Microbiology Antigen/Antisera Shigella ()
ANTISERA, TOXOIDS, VACCINES AND TUBERCULINS IN PROPHYLAXIS AND TREATMENT.

Universal flu vaccine

Universal flu vaccine tests start

Prof. Adrian Hill explains the vaccine.
A universal flu vaccine which could mean an end to the annual jab is being tested on UK volunteers.
It targets a different part of the virus to current vaccines, which means it does not have to be altered every year to match circulating strains.
If successful, the vaccine developed by Oxford University researchers would also be a key weapon in a flu pandemic.
Experts said such a vaccine was the "Holy Grail" for flu researchers but there was still a long way to go.
Study leader, Dr Sarah Gilbert, said traditional influenza vaccines are designed to prompt an immune response to H and N proteins on the outer shell of the virus. But these proteins are prone to mutation - and every year the vaccine has to be reformulated on the basis of the strains likely to be most prominent.
So instead, the researchers have developed a vaccine on the basis of proteins inside the cell, which are far more similar across different strains. The vaccine uses a weakened smallpox virus to carry the proteins into the body - a technique that has already used in malaria and TB vaccines. Once the virus has invaded the cell and starts to multiply, these inner proteins called matrix protein 1 and nucleo - protein, are revealed to the immune system. A specific type of immune cell, called a T cell, then learns to recognize and destroy cells containing the proteins the next time it encounters them.

Tests
Initially 12 people will be vaccinated to test the dose before further studies are done to check its effectiveness in people exposed to flu.

How flu vaccine works
Step 1- New universal flu vaccine is injected into the arm and is taken up by healthy cells.
Step 2- Cells containing vaccine attract immune cells which multiply and move around the body.
Step 3- Immune cells now trained to recognize proteins inside virus, which enters body via airways.
Step 4- Killer immune cells recognize flu-infected cells and destroy them along with flu virus.

Dr Gilbert said if they were successful it could drastically change the way flu vaccine is used.
"With having to make a new vaccine every year there's never enough to go around. With this vaccine, we could end up having pretty much everyone vaccined - a situation more like measles where you don't really see it anymore"
In the case of a pandemic, stockpiles of the vaccine could be made in advance instead of having to wait for an outbreak to then identify the particular strain of flu.
Potentially, once people had received the vaccine they would only need a booster once every 5 to 10 years. But she added the research team had 5 to 10 years of further tests ahead of them. However, it is hoped a similar approach might eventually also be used to combat HIV, TB, malaria and even cancer.

Professor John Oxford, a flu vaccine expert at Queen Mary, University of London said such a vaccine would be the "ultimate prize". But it's a fairly difficult prize to get - it may just be a question of luck. "There are people trying all kinds of strategies."
He added that having to manufacture different flu vaccines every year was a "huge burden" on pharmaceutical companies. "This team has experience with this type of vaccine so they may well get there."

By Emma Wilkinson
Health reporter, BBC News

 
Merck Mmrii Vaccine 0.5Ml Measles Mumps And Rubella - Box of 10 - Model 00000468100

E-test pic

Determining MIC by E-test