Lab 5 Instructions

EVALUATION OF ANTIMICROBIALS FROM LAST LAB PERIOD

Chapter 24: Chemical Methods of Control, Disinfectants and Antiseptics

Last week, each lab group had three Petri dishes with nutrient agar.

One dish has bacteria (Pseudomonas or Staph aureus) that were exposed to Mouthwash. One dish was exposed to Pine-sol, and one was exposed to contact lens cleaning solution. Record your results at the end of Chapter 24 in the lab manual. How many minutes did it take for each product to kill all the bacteria? Which disinfectant worked best? How do you know?

Chapter 25 (p. 191)

Each lab group took 3 Petri plates of Mueller-Hinton agar (looks like nutrient agar). You inoculated one plate with Staph aureus (Gram +), one with E. coli, and one with Pseudomonas aeruginosa (both Gram neg) by streaking for confluence. You put 5 different antibiotic discs and set them on each plate. Today, measure the zone of inhibition (from the EDGE of the disc to the outer boundary of the clear area with no bacterial growth; record in mm) of each colony and record them in the back of Chapter 25 of your lab manual. Which of your bacteria were sensitive to which antibiotics? Which bacteria were resistant to which antibiotics? Which have intermediate sensitivity? Before you can answer these questions, I need to explain how to determine sensitivity, resistance, and intermediate sensitivity. Then write your results in your lab manual.

Explanation

Antimicrobials are either narrow spectrum or broad spectrum. NARROW SPECTRUM antimicrobials are effective against Gram positive organisms because their mechanism of action is as a cell wall inhibitor, which does not cause a problem for Gram negative organisms. BROAD SPECTRUM antimicrobials kill both Gram positive and Gram negative organisms because the part of the cell that they damage (e.g. ribosomes) is present in both types of organisms. However, you need to be careful with broad spectrum antimicrobials, because if there are a lot of Gram negative bacteria in the blood stream and you kill them, their endotoxins will be released into the blood stream, which can be deadly to the patient.

Today we will measure the zone of inhibition, which is the clear zone around the disc, which has no bacterial growth. Each laboratory uses the same media and antibiotic discs, and the size of each disc is always the same. Therefore, this experiment is standardized and you can use an antibiotic sensitivity table (Kirby-Bauer chart) to evaluate your results. Because the discs are the same size, you measure from the edge of the disc to the edge of the clear zone. Do NOT measure the whole diameter of the clear zone. The zone of inhibition determines the effectiveness of the antimicrobials ONLY when checked against the Kirby-Bauer chart (p. 3 of this document). Just because one zone is the largest, it does NOT mean that is the most effective antibiotic.

Below is an example of an antibiotic sensitivity table. The actual chart is in your lab manual (p.193).

Resistant / Intermediate / Sensitive
Ampicillin / <12 / 13-18 / >19
Amoxicillin / <17 / 18-20 / >21
Cephalexin / <16 / 17-19 / >20
Cephadroxil / <20 / 21-24 / >25

Suppose you measure all the zones of inhibition on your plate, and the largest one measures 20 mm. Is that the antibiotic that is most effective? NO! We cannot answer that question until we check the chart. According to this sample chart, if the zone of inhibition for your Cephadroxil disc was 20 mm, the organism is resistant to that antibiotic, and you should NOT prescribe Cephadroxil to that patient. The zone for Cephadroxil needs to be 25 mm or more to be effective. If the zone of inhibition for your Ampicillin disc was 19 mm, check the table and see that the organism IS sensitive to Ampicillin, so that is the antibiotic that you give the patient. Therefore, the antibiotic with the largest zone of inhibition is NOT necessarily the most effective one. Intermediate sensitivity implies clinical efficacy only in body sites where the drugs are physiologically concentrated (e.g. quinolones and beta-lactams in urine) or when a higher than normal dosage of a drug can be used. Choosing an antibiotic in the “sensitive” category is the best choice. However, if the patient is allergic to that medicine, choosing TWO antibiotics in the “intermediate” category is the next best option, because they give a synergistic effect. One might inhibit protein synthesis, while the other interferes with the cell wall structure. Alone, they cannot kill the organism well, but together they are effective. The first treatment option for the patient is an antibiotic that is in the sensitive category. If that is not possible (because there are no such drugs for that organism, or the patient is allergic to the drug), the second treatment option is to give two antibiotics that are in the intermediate category (combination therapy), because they have a synergistic effect.

Chapter 26 (Hand scrubbing)

Each LAB GROUP gets two NA plates.

Take one NA plate and divide it into 4 quadrants and number them with small numbers in the corners. Label the plate with Magrann, date, your name, and “water”.

Take a second NA plate and divide it into 5 sections. Label the plate with Magrann, date, your name, and “soap”.

On the water plate, touch section 1 with your fingers. Wash well with a scrub brush without soap, shake off the excess water, and while your hands are still wet, touch section 2. Do not dry your fingers. Wash again, and while your hands are still wet, touch section 3. Wash a final time, and touch section 4. Use the same fingers to touch each quadrant.

On the soap plate, use the same hand. Use a scrub brush to wash well with soap, rinse, shake off the water, and touch section 1. Wash again with soap, rinse, shake of the water and touch section 2. Using a brush and soap, scrub your hand for 2 minutes, rinse, and shake off the water and touch section 3. Repeat the soap and brush scrub for 4 minutes, rinse, and shake off the water, then touch section 4. Use a waterless hand-cleansing product, then touch section 5. What are the active ingredients in this product?

They go in the plastic Petri dish sleeve, inverted. Incubate the plates at body temperature.

Chapter 45: Skin Bacteria

The dry keratin layers of the epidermis of the skin are not easily colonized by most microbes. Sebum from oil gland and salts in perspiration inhibit the growth of most microbes. Therefore, the skin is an inhospitable environment for most microbes. Only a few types of bacteria (normal resident microbiota) can tolerate the dry keratin and the hypertonic environment of the skin (which is caused by salt in our perspiration). The most common types of bacteria found on the skin are Gram positive, salt-tolerant organisms such as Staphylococcus aureus. More bacteria are found in moist areas, such as the axilla and the sides of the nose. Transient microbes may be present on the hands and arms because of contact with the environment, but they do not live on our skin for long. Propionibacterium live in hair follicles on sebum produced by our oil glands. The propionic acid they produce maintains the acidic pH of our skin (pH 3-5), which suppresses the growth of other bacteria. Streptococcus is also part of the normal microbiota of the skin. Mannitol salt agar (MSA) is selective for salt-tolerant organisms and it is also considered to be a differential media because mannitol-fermenting organisms will produce acid, turning the pH indicator in the media yellow. Staphylococcus aureus is part of the normal microbiota of the skin and is a pathogen. If the skin becomes broken, it can invade and cause disease. Both pathogenic and non-pathogenic species of Staphylococcus produce catalase and collagenase, and they are all oxidase negative. Staph aureus makes an enzyme called coagulase, which clots the blood (clumping), whereas other species of Staphylococcus do not produce this enzyme and are not pathogenic. If MSA grows an organism, we know it is Staphylococcus, but then we do a coagulase test to see if it is Staphylococcus aureus or not.

Procedure

Each GROUP takes one Mannitol Salt Agar (MSA) plate and inoculates it with a swab from your own skin surface by streaking for confluence (tight zig-zag from top to bottom) with a cotton swab. Moisten the sterile swab with plain water, and swab an area of your skin. Good areas to find Staph aureus include the outside of your nose, the inside of your nose, the axilla (armpit), and the inside of your elbow. However, you can swab wherever you like. Write down the location you chose in your lab manual at the end of Ch 45. Then add a Novobiocin antibiotic disc in the center.

After your MSA colonies grow, next week, each INDIVIDUAL takes one API Staph strip and inoculate it with a colony from your MSA.

Chapter 46: Respiratory Bacteria

The lower respiratory tract (larynx, trachea, bronchial tubes, and alveoli) is normally sterile, but the upper respiratory tract (nose and throat) is not because it is in contact with the contaminated air we breathe. The throat is a warm, moist environment, allowing many bacteria to thrive. It is normal to find Staphylococcus, Streptococcus, Neisseria, and Haemophilus as normal microbiota in the throat. However, we do not often become ill because of their antagonism toward each other. They suppress each other’s growth by competing for nutrients and producing inhibitory substances. Streptococcal species are the predominant organisms in throat cultures, and some species are the major cause of sore throats. Pathogenic bacteria may produce hemolysins, which breaks down red blood cells.

HEMOLYSIS TEST (Controls: Beta = Strep pyogenese; gamma = E. coli, alpha = Streptococcus pneumoniae)

You will use a throat swab to inoculate a blood agar plate and use this plate to observe hemolysis patterns. Beta hemolysis (Streptococcus pyogenes, Staphylococcus aureus) means the organism can completely lyse red blood cells and they digest the hemoglobin (they are pathogenic bacteria), so there will be clear zone with a clean edge around the colonies on your plate. Alpha hemolysis (Streptococcus pneumoniae) means the bacteria partially destroys the red blood cells, and they oxidize the iron in the hemoglobin, which turns the colony green, with a cloudy zone around the colony and NO clear areas. Gamma hemolysis means the organism is non-hemolytic, so there will be NO clear areas, and the colony will not be green.

Streptococci that are alpha or gamma hemolytic are usually normal microbiota, whereas beta-hemolytic streptococci are usually pathogens. More than 90% of streptococcal infections are caused by Beta-hemolytic Group A Streptococcus pyogenes, which is sensitive to the antibiotic bacitracin, whereas other streptococci are resistant to it.

Procedure

Each INDIVIDUAL takes one Blood Agar plate and labels it with the date, Throat swab, your name, and Dr. Magrann. Have your lab partner take a sterile cotton swab and touch it to your throat at the location shown Ch 46 of your lab manual. Do NOT swab your own throat. Make sure you do not touch the cotton to the tongue. Do not wet it with water first; use it dry. Then use the swab to make a loose zig-zag streak onto the blood agar plate. On Saturday, look for alpha (green zone), beta (clear zone), and gamma (no change) hemolysis. For a demo, the lab tech will inoculate one blood agar plate with Streptococcus pyogenes on one half and Streptococcus pneumoniae on the other half. This will be a control plate for the whole class to observe.

When you have inoculated all the plates from Chapters 25, 45 and 46, tape together all of the plates from your lab group, label the tape with the date, Magrann, Ex 26, 45, 46, and your lab group leader name. and place them INVERTED (agar on the top) into the incubator.

SOIL PROJECT

Examine your streak for isolation plates. Use a needle to pick an isolated colony and streak a slant. Select another isolated colony and streak another slant. Label the slants as Magrann, your name, date, “Soil”, and your choice of name for the organism. After 2 days of room temperature, keep these in the refrigerator as a reserve. Run all of your biochemical tests from the Master plate for the rest of the semester.

Today, do a Gram stain on your organism from your streak for isolation plate, plus perform the lab experiment tests on it as we go through the rest of the semester.

Use your pure culture to perform the following tests for morphological characteristics

1)  Gram stain to determine Gram +/- and shape (rods or cocci)

2)  Negative stain to determine shape and arrangements (staph, strep, diplo, singles)

Use your pure culture to perform the following tests for cultural characteristics

1)  Inoculate a broth to observe colony morphology in a liquid next lab.

2)  Determine if your organism displays hemolysis by inoculating a blood agar plate. Label it with “soil” and the other data so it does not get mixed up with your body swab on blood agar.

NEXT TIME, CHECK YOUR PLATES FOR THESE THINGS:

Chapter 26 (Hand scrubbing)

You had two plates of NA divided into quadrants; one was labeled “water” and the other was “soap”. Record the growth in each quadrant on each plate as negative (no growth), + (minimum growth), 2+ (moderate growth), 3+ (heavy growth) and 4+ (maximum growth. How do resident microbiota and transient microbiota differ? Which quadrants contained resident microbiota, and which contained transient? What is the purpose of a surgeon scrubbing with an antiseptic and a brush for 10 minutes?