Lab 30: Staphylococcus aureus Rapid Test

Lab 30: Staphylococcus aureus  Rapid Test 

1. Rapid Tests= immunological assays that use Ag-Ab rxns to identify a specific organism or agent

-give results within minutes and can be run in hospitals labs or dr. offices

-In this lab, we do Passive Agglutination Test for Staphylococcus aureus

Passive Agglutination Test= uses latex beads coated with anti-Staph antibodies to detect S. aureus

-test kit comes with positive and negative controls 

-test samples come from beta hemolytic colonies cultured from throat swabs grown on sheep’s blood agar plates

+= if S.aureus antigen is present and binds to Abs= agglutination = clumping

-= no clumping

-Indications of Positive Test

a. A Positive test combined with appropriate signs/symptoms can confirm dx of S.aureus infection

S. aureus= Gram + non motile coccus

-responsible for wide array of diseases including:

a. skin infections= most common

-including: pimples, impetigo, boils, carbuncles, and cellulitis

b. surgical and would infections are common and can spread to internal organs causing other disease like pneumonia or septicemia

c. Staphylococcal food poisoning and toxic shock syndrome from toxins produced from certain strains of Staph

b. Can indicate that person is a healthy carrier= healthy, asymptomatic carrier of S. aureus  in their noses/throats or skin, but NO symptoms of disease

-25-50% of population =health carriers of S. aureus 

-healthy carriers can still spread Staph to others 

-even resistant strains of Staph like MRSA (methicillin-resistant S. aureus) can be found in healthy carriers

- important for healthcare workers who are healthy carriers but are alsoaround immunocompromised patients- could be very harmful if spread to them

Wells 4,5, 10= positive =clumping

Well 9= negative= no clumping

Lab 34: Throat Culture

Lab 34: Throat Culture 

1.  Pharynx=Throat= warm, moist environment that houses a mixed normal flora with organisms from both the upper respiratory and digestive tracts

-besides normal flora, the pharynx can also harbor potentially pathogenic bacteria

-these pathogens are kept in check by normal flora, but they can cause disease if they breach this barrier or      are transmitted to vulnerable individuals 

-In this lab, we take throat swabs from nasopharynx, which is the respiratory part of the pharynx (in the far back region of throat, behind uvula) 

2. Growth medium= sheep’s blood agar plates-contains 5% sheep’s blood, and is a differential medium agar which helps to categorize bacteria by hemolysis patterns -=how bacteria destroy RBCs (hemo- RBC; -lysis= burst)

ie. differential medium agar for hemolysis patterns

3. Hemolysins=toxins produced by colonies that cause either partial or dotal destruction of surrounding RBCs

4. We see Three Hemolysis Patterns:

1. Alpha (α) Hemolysis= partial clearing and greenish pigmentation around colonies

a. Alpha hemolytic bacteria:

1. are normal flora

2. MOST COMMON bacteria in throat

2. Beta (β) Hemolysis= complete lysis of RBC around colonies resulting in a clearing 

a. Beta hemolytic bacteria:

1. usually pathogens 

ex. Streptococcus pyogenes

      Staphylococcus aureus 

3. Gamma (ɣ) hemolysis= no clearing or surrounding ring, just dark colony with sheep’s blood agar surrounding it

a. Gamma hemolytic bacteria= normal fora 

Colonies on upper right are Alpha Hemolytic 

 

Mix of Alpha, Beta, and Gamma 

Mix of Alpha, Beta, and Gamma

Pure  Beta hemolytic bacteria

Pure Beta Hemolytic bacteria- notice the huge clearing 

Lab 29: Quantitative ELISA

ELISA (Enzyme-linked immunosorbent assay)= an immunodiagnostic test used to measure specific antigens or antibodies in an unknown sample 

-test is often used to detect the presence or even specific levels of substances such as drugs, hormones, foods, and pathogens

-test is relatively inexpensive, simple, and easily interpreted 

-each test is run in a small plastic well on a microtiter plate= plate with multiple wells for test

-test works bc of antigen-antibody specificity 

-Testing for Unknown Antigen= the unknown antigen will adhere to its specific antibody in the test well 

-Testing for Unknown Antibodies= the unknown antibody will adhere to its specific antigen in the test well 

*in both types of tests an antibody- enzyme conjugate is added

Antibody-enzyme conjugate= another antibody that is linked to an enzyme 

-this enzyme catalyzes a color change rxn in the presence of its substrate, a chromagen 

-chromogen= chemical that can undergo a color change rxn 

-2 Types of Info Obtained from ELISA

1. Qualitative ELISA test=  a color change is enough to confirm a positive result 

Ex. Pregnancy test, or peanut contamination in food- these are yes/no results

2.  Quantitative ELISA test=  the level of the substance is also assessed 

-to do this, the intensity of the color change is read with a spectrophotometer=gives an absorbance reading which is proportional to the amount of antigen or antibody

Ie. High absorbance= high amount of Ag or Ab 

-the results are then compared to absorbance readings from known amounts of Ag or Ab

- we then plot these known values on a graph called a standard curve to be able to determine the level of unknown from the graph

-2 Types of ELISA tests: Direct and Indirect

1. Direct ELISA= “sandwich assay”= tests for an unknown Antigen 

a. well is coated with known Antibody for the specific antigen to be tested 

b. sample is added with unknown antigen, and if present antigen will bind to Antibody 

c. Wash= any unbound material is then washed away 

d.  Antibody-enzyme conjugate is added

e. Wash- unbound material is again washed away 

f. Chromogen added 

color change= positive test= yes Ag 

no color change= negative= no Ag

-sandwich assay- bc it creates a symmetrical grouping of Ab-Ag-Ab

Ex. Direct ELISA= pregnancy test, influenza test 

2. Indirect ELISA= tests for an unknown Antibody 

a. Well coated with known Antigen for the specific antibody to be tested

b. Sample is added – if antibody present will bind to Ag

c. Wash- unbound material is washed away 

d. Antibody-enzyme conjugate added

-this is actually an anti-antibody, bc it adheres to the specific class of Ab that we are testing for 

e. Wash- unbound material washed away

f. Chromogen added

positive= color change

negative= no color change 

ex. Indirect ELISA= HIV antibodies-> bc HIV antigens hide in infected cells and can be harder to find in serum samples 

In this lab- we do indirect ELISA test for salivary IgA antibodies 

IgA= predominant secretory Ab (ie. Found in secretions-saliva, tears)

• IgA can inhibit bacteria that contribute to dental caries= cavities
• Prediction= increased IgAs= decreased cavities bc it inhibits bacteria that cause cavities

*Note- that for our indirect ELISA- the test wells have anti-human IgA adhered to them – this is our Ag

-this is known as a capture antibody- acts like an Ag for Ab- we got by injecting into goats and collecting the Abs made by goat

Brief Outline of our Steps- we do a Indirect Quantitative ELISA for Salivary IgA

1. Add saliva sample which contains IgA to test wells with anti-human IgA adhered to them 
2. Wash
3. Add anti-human IgA Ab conjugated to the enzyme horseradish peroxidase (HRP)
4. Wash
5. Add TMB chromogen= HRP enzyme substrate-

ie if HRP is bound and present TMB chromogen will cause it to change color

Color change should occur- then we place microtiter plate into the ELISA plate reader= spectrophotometer=determines the absorbance readings 

Determine amount of salivary IgA- we do this by creating a standard curve of absorbance readings of known concentrations of human IgA (ie. We graph standards of known concentrations and their  known absorbance’s)

For Graph= makes it a quantitative

X=concentration of IgA standards

Y= absorbance (from spectrophotometer) 

Lab 33: Simulated Epidemic

Lab 33: Simulated Epidemic

1. Epidemic (level)=  the incidence of a disease in a population at greater than expected levels 

2. Endemic (level)= the expected low level of a disease  

-disease can transition from endemic to epidemic levels for a variety of reasons 

3.Pandemic=  when a disease travels globally and affects people on several continents

ex. influenza, AIDS

4. Epidemiology= field of science dedicated to studying the location and timing of diseases and how they spread

-using this info, epidemiologists can gather data to better understand diseases, in the hopes of controlling or preventing them

-one specific job they have is to find source of disease outbreaks, can be common sources like contaminated food, water, or a person

5. Index Case=  the first person to have a disease which has lead to an outbreak 

-In this lab, we simulated spread of an Epidemic by direct contact- handshaking, and tried to find index case 
--during data collection only half of the class cultured after the first round of handshaking- we did this bc, during the beginning of an outbreak it is rare for Epidemiologists to have all cases 

-Micrococcus luteus= bacteria used in our “outbreak”

       -grows yellow colonies when grown on agar 

Plate1 (on left)= Positive for Micrococcus luteus- its yellow

Plate 9 (on right) = Negative -small random colonies (not yellow)

Closer look at Micrococcus luteus

Lab 27 Identifying GM Crops

Two methods currently used to identify GM genomes

    1. Enzyme Linked Immunosorbent Assay (ELISA)

    2. Polymerase Chain Reaction (PCR) 

             -things needed for PCR: template DNA, nucleotides (ATCG), DNA Primer, DNA polymerase

Common regulatory sequences used to control GM Genes which include: 

   1. Promoter=35S promotor from cauliflower mosaic virus 

   2. Terminator= Nopaline synthase (NOS) terminator from Agrobacterium tumefaciens

ie. Promotor---gene –terminator 

- PCR checks that we have plant DNA by detecting PSII chloroplast gene

-then checks for GM by seeing if DNA has 35S promotor and NOS terminator 

-if yes (ie. Band) to both then food is GMO

-if no plant, then no conclusions can be made-must repeat test

-if no GMO DNA then not its not genetically modified 

We used PCR to detect 35 promoter, NOS terminator, and PSII chloroplast gene

 Then we used Electrophoresis to visualize DNA fragments 

 Machine Used to Run Current Through Gel   

Gel of GM Food Negative        

                                                                                          

Lab 32: Antibiotic Sensitivity

 Antibiotic= a natural substance (made by fungi or bacteria) that inhibits or kills microorganisms

-in common usage antibiotics are often equated with antibacterial drugs and synthetic drugs

      --in 1928, Alexander Fleming found a mold contaminant on an agar that inhibited growth of Staphylococcus aureus

-the mold was Penicillium chrysogenum and so the inhibitory chemical was named Penicillin

        -both Penicillin and Sulfas were developed during WWII, with derivatives also being developed 

         -antibiotics were thought to be the "magic bullet" to wipe out bacterial infections

         - However, bacteria are becoming resistant to traditional antibiotics 

Bacteria develop resistance through:

1. mutation 

2. acquisition of R plasmids 

        -resistance promoted from the misuse/overuse of antibiotics

Resistant Bacteria=

-S. aureus (from Flemings lab) is now major resistant bacteria 

-MRSA=methicillin resistant S. aureus

-VRSA= Vancomycin resistant S. aureus

       -Increased bacterial resistance, means that more antibiotics are needed, but drug companies don't want to produce        antibiotics because they are too expensive to make

                 -can take up to 10 years to make: must find, test, get approved, produce

                 -and after that bacteria can still develop resistance to drug

3 ways to treat infections

1. Trial and error approach= patient given a drug that may or may not work

2. giving them lots of antibiotics- ie kitchen sick= promotes drug resistance and has many side effects to          patient

3. Determining susceptibility is the best way to give patients the proper drugs in the proper dosage

Ways to Determine Susceptibility

1. Minimum Inhibitory Concentration (MIC)= the minimal concentration one is needed to inhibit growth of bacterium

used in:

a. Broth dilution methods-fill test wells with increasing concentration of antibioitic and with the same      concentration of bacterium, then look for turbidity, 1st well with no cloudiness= MIC

b. Kirby-Bauer Disk DIffusion test= small disks with standard concentrations of antibiotics are placed on agar with bacterium

-the antibiotics diffuse outward, creating a zone of inhibition (no growth) around disks

-the size of the zone cannot be directly compared due to the variable solubility and diffusion rates of the  drugs in the medium, so zones must be compared to a standardized chart to assess if the bacteria are sensitive or resistant

-ie. only tells you if bacteria is sensitive or resistant 

c. E test- small strips with quantified antibiotic gradients are used

-zones of inhibition form a teardrop (since high concentrations will diffuse farther than low concentrations)

-the smallest part of teardrop=MIC

-advantage of this test is that it is quantitative for MIC

In this Experiment- test selected antibiotics using the Kirby Bauer Disk Diffusion Test 

-ie. tells you if bacteria is sensitive/resistant and correct dose

-once we grew plates, we looked for zones of inhibition 

-then using a transparency, we overlaid the appropriate antibiotic disk diagram onto its antibiotic disk to determine if the bacteria are Sensitive (S), Intermediate (I) or Resistant (R) to each antibiotic

- The bacteria are S,I, or R if they are in the S,I, or R zone 

R-Resistant= Zone of inhibition with a diameter equal to or less than that of the inner circle

I- Intermediate=Zone of inhibition with a diameter greater than R, but less than that of the outer circle

S- Sensitive= Zone of inhibition with a diameter equal to or greater than that of the outer circle 

****note- Colonies growing WITHIN the zone of inhibition = bacterium resistant to antibiotic (Yikes!)

 -We had two plates

- antibiotic disks with NO ZOI are all Resistant to antibiotic

-so for #33 thats all but one disk 

Plate #16

 Sensitive

 Sensitive 

Sensitive

Resistant 

(I think some got Intermediate on this one)

 Plate #33 =Sensitive 

Plague Fighters

Plague Fighters

1. What was the cause of the epidemic? 

-Ebola= virus

2. Where and when did this epidemic occur? 

-Kikwit in Democratic Republic of Congo (Africa) in 1995 

3. What are the symptoms? 

-attacks vital organs- results in bleeding inside and out, rash, vomiting blood, eyes filled with blood, fixed stares (frozen facial expression), death after 10 days

4. How is it transmitted? 

-coming into direct contact with bodily fluids: tears, blood, surface of skin, sperm

5. How did the scientists prevent the spread of the epidemic?

- Found every case and isolated them ie. quarantine 

-used protective clothing to prevent spread and bleach to kill virus

-stopped ritual funerals where dead person was touched, instead dead were immediately body bagged and buried (no touching)

6. What is meant by “chain of death”?

- you could see the progression of primary care givers of the sick becoming sick

7. What is the natural host for the disease?

-unknown

8. Who was the index case for this outbreak, and what did he do for a living?

-Gaspar Menga- charcoal worker from Kikwit

9. What is the treatment for Ebola?

-None, just supportive care

10. How was nurse Nicole treated? Did she recover?

-blood transfusion from a survivor of Ebola, it contains antibodies against Ebola

11. What was the controversy surrounding her treatment?

- she could obtain other diseases the blood donor has like HIV, Hepatitis, Malaria

-they could have misdiagnosed her with Ebola and actually give her Ebola 

12. Could there be alternate reasons that the treatment was successful for the patients seen at the end of the epidemic?

-after going through many people- virus is less virulent at end

-late in epidemic people were getting smaller viral loads when infected- so didn’t get the virus as badly

13. What is the major concern about highly virulent diseases like Ebola (think about the Reston, Virginia, incident)?

-that they will be airborne

14. Why might Ebola outbreaks become more common in the future?

-bc people are moving into rain forests as populations expand

- brings us closer to whatever the source of Ebola is