this zip file has everything needed. Please dont have plagiarism strictly, answer the questions using the video and please complete it. All i need is student template the api problem and diffuion problem i think
Microsoft Word - API Test Strip Results.docx API Test Strip Results API test strip standards Use the below standards as a guide to determine positive and negative tests. Identify the species by entering the +/- data at https://apiweb.biomerieux.com/. User name: aadolfsson Password: biol321 Negative standard: Positive standard: Results from video ‘Identifying Microbes’ (Escherichia coli) Note: Oxidation test result (21st test) is -ve API test strip results from unidentified infections 1) Patient sample (real image) Note: Oxidation test result (21st test) is -ve Note: The “VP” test solution (test 10) is actually pink 2) Patient sample (hypothetical) Note: Oxidation test result (21st test) is +ve 3) Patient sample (hypothetical) Note: Oxidation test result (21st test) is -ve CHEM200 Living in a Molecular World PRACTICAL MANUAL School of Chemistry and Molecular Bioscience University of Wollongong 2024 Name: Demonstrator: 2 LABORATORY SAFETY GUIDELINES • Attend practical class on time in order to receive important instructions. • It is your responsibility to listen and follow instructions. • Lockers are to be used for storage of bags, jackets, etc. during the practical class. This includes your phone! Phones are not to be used as calculators in the laboratory. • You will not be allowed in the lab unless you are wearing enclosed shoes. No thongs, open-toed sandals or bare feet – see the poster on the lab door for more details. • Familiarise yourself with the location of all fire extinguishers, fire blankets, safety showers and emergency exits. • Common sense is expected at all times. There should be no horseplay or practical jokes in the laboratory. • No drinking, eating, chewing gum, or applying lip balm in the laboratory. • You must wear a lab coat and safety glasses at all times. • Before leaving the laboratory, you must always remove your lab coat and wash your hands. • You will be advised of any hazards that require your attention either verbally or in the lab manual. Safety data sheets (SDS) will be provided for any chemicals that pose a risk. • Keep your workspace as tidy as possible throughout the practical e.g. discard or return materials/equipment as soon as you are finished with them. • Do not work on top of your lab manual; keep it to the side or to the back of your work area. • All microbiological cultures should be treated as potentially pathogenic and handled with care. Always use aseptic technique (see next page). • Follow instructions for disposal of all waste, including contaminated waste and broken glass. If in doubt, ask a demonstrator or the lab supervisor. • Wash your hands immediately and thoroughly if they are contaminated with microorganisms or any chemical reagents. • Spills can happen; how you deal with them is of importance! Report any spills IMMEDIATELY to a demonstrator or the lab supervisor. They will advise you on the correct clean up procedure or clean up the spill for you. • Report any accidents, no matter how trivial, to a demonstrator or the lab supervisor. 3 ASEPTIC TECHNIQUE Aseptic technique is a set of specific practices and procedures designed to minimize or prevent contamination by microorganisms. This includes: • protecting yourself or others from infection, and • protecting the culture that you are working with from contamination. The main sources of contamination are: • air (contains dust particles), • laboratory surfaces, • hands, • clothing, and • breath/saliva. In these practicals you will maintain aseptic technique by following these simple guidelines: • Disinfect work areas before and after any microbiology work. • Use sterile equipment. • Do not allow anything sterile to touch anything non-sterile (except as necessary for the procedure). • Keep your hands clean during procedures and wash them when you are finished. • Open containers for as short a time as possible. • Keep loosened lids on containers, don’t place them on the bench. • Avoid talking while preparing and inoculating media. • Decontaminate any biological spills immediately. • Appropriately dispose of all contaminated items in the waste bins provided. 4 INTRODUCTION TO MICROBIOLOGY Microbiology is the study of microscopic organisms (called microorganisms or microbes). In these practicals you will culture (grow) microorganisms from environmental and laboratory samples. Like us, microorganisms need specific conditions for growth and each has their own requirements for humidity, gas, nutrients, and temperature. In our experiments we will only be controlling the temperature and nutrient availability. The nutrient mixtures used to grow microorganisms in the lab are called media (singular medium). They usually contain carbon and nitrogen sources (e.g. sugars and amino acids), salts, and vitamins. Media can be liquid, solid or semi-solid. In these experiments we will be culturing samples on agar plates (Figure 1). These are petri dishes filled with media that has been solidified using agar (a compound derived from algae). The medium that we will use is tryptic soy agar (TSA), a general purpose medium made from soybean, milk protein, sodium chloride (table salt), and agar. Microbes grow on agar as either colonies or lawns (Figure 2). A colony is a visible mass that is assumed to have grown from a single cell. A lawn is an even covering of growth that occurs when colonies are so close together that they overlap. The pattern of growth will depend on how the agar plate is inoculated. In microbiology, inoculate means to introduce microorganisms into a culture medium. In these experiments you will inoculate plates using either a transfer pipette and cell spreader, an inoculation loop, or a cotton swab (Figure 3). These are commonly used microbiology tools that come in various sizes, shapes and materials. You will be using disposable versions that are already sterile (meaning they don’t contain any living organisms). TIPS FOR USING AGAR PLATES Label plates before you inoculate them. Always write on the part which contains the agar (not the lid!), around the edges as this makes it easier to see your results. NEVER write on the agar gel itself! The media is not completely solid (more like jelly) and it’s easy to damage the surface. Apply gentle pressure and when possible, approach the surface at an angle instead of head-on (Figure 4). Figure 1. Agar plate. Figure 3. Common microbiology equipment. Figure 2. Microbial growth. Figure 4. Using agar plates. 5 PRACTICAL 1. MICROBES IN THE ENVIRONMENT Microorganisms are found almost everywhere on Earth: in air, soil, and water, as well as on the outer and inner surfaces of our bodies (and those of other animals). Microbes are vital to the planet’s ecosystems, for example through their roles in decomposition, oxygen production, and nitrogen fixation. Some microorganisms cause diseases but others are important to human health. Microbes in your gut help digestion, and also play a role in avoiding disease. Many microbes have been “domesticated” and are used to produce food (e.g. yoghurt, cheese, beer, bread) or pharmaceuticals. In today’s practical you will inoculate tryptic soy agar (TSA) plates with environmental samples, in order to examine the diversity of microbes found in your local environment. The plates will be incubated at 25°C until next week’s practical, when you will get to observe your results. MATERIALS At your bench • Fresh TSA plates • A marker for labelling plates and tubes • Sterile micro tubes • Sterile 50 mL tubes (orange caps) • Sterile transfer pipettes • Sterile cell spreaders • Sterile saline (salt water) At other stations around the lab • Pond water • Soil mixed with sterile saline WORK IN GROUPS OF FOUR 1) Label (see notes below) your six agar plates with the name of the sample, the date, and something to identify your group (e.g. air exposure 30/10/2017 science wizards). Note: − Remember to label the agar side of the plate, not the lid and not on the agar gel. − Write as small as possible and around the edges (so that you have a good view of what grows!). Table 1. Plate Sample name Inoculation 1 air exposure exposed to the air for 1 hour 2 tap water 5 drops of tap water 3 pond water (1/5) 1 drop from tube “P 1/5” 4 pond water (1/25) 1 drop from tube “P 1/25” 5 soil (1/100) 1 drop from tube “S 1/100” 6 soil (1/1000) 1 drop from tube “S 1/1000” 2) Take the lid off the air exposure plate and leave it open on the bench with the agar surface facing up. Note the time. Replace the lid after 1 hour. 3) In three separate tubes (remember to label them first!) collect approximately 5 mL of tap water, 5 mL of pond water, and 5 mL of soil + saline. 6 SERIAL DILUTIONS Many experiments don’t give useful results if the starting sample is too concentrated. We need to first make a series of diluted samples and test each one. Figure 5 shows how to make the serial dilutions that you will use to inoculate your plates. Figure 5. Making serial dilutions. 4) Make dilutions of your pond water and soil water samples (see the explanation above): − Label five separate micro tubes: “P 1/5”, “P 1/25”, “S 1/10”, “S 1/100”, and “S 1/1000” − Using a sterile transfer pipette, add four drops of saline to each of the “P” micro tubes and nine drops of saline to each of the “S” micro tubes. − Using a new transfer pipette, add one drop of pond water to “P 1/5” and mix. Then add one drop from “P 1/5” into “P 1/25” and mix. − Using a new transfer pipette, add one drop of soil water to “S 1/10” and mix. Then add one drop from “S 1/10” into “S 1/100” and mix. Then add one drop from “S 1/100” into “S 1/1000” and mix. 5) Inoculate plates 2, 3, 4, and 5 using the volumes given in Table 1. Inoculate the plates one at a time, using a new transfer pipette and cell spreader each time. − Drop the liquid into the centre of the plate with a transfer pipette. − Use a cell spreader to spread the liquid over the surface of the agar. Move the spreader back and forth over the entire surface, turn the plate about 90 degrees, then cover the surface again. Repeat this until you’ve covered the entire surface four times. 6) Tape the agar plates closed by wrapping a parafilm strip around the edge of the lid. You will need to cut the supplied parafilm into six strips (one for each plate), each one being half a square wide – consult your demonstrator if you are unsure. 7) Place your agar plates into the collection tub, ensuring the agar side is facing up (lid facing down) as this prevents contamination from condensed water droplets falling onto the agar. You should have six plates in total. CLEAN UP (PRAC 1) Wipe up any spills with paper towel. Put the dirty paper towel in the normal bin at the back of the lab. Empty the tap water, pond water, and soil + saline down the sink and flush with more tap water. Put transfer pipettes, cell spreaders and packaging in the biohazard waste containers on your bench. 7 PRACTICAL 2. ANTIBIOTICS, ANTIFUNGALS AND ANTISEPTICS Antimicrobial drugs are chemicals which kill or inhibit the growth of microorganisms. In this practical you will use chemicals from three different groups of antimicrobials. Antiseptics are applied to living skin/tissue, and are active against a range of microbes including bacteria, fungi and viruses. Microorganisms are very diverse, however, and so not all antimicrobials are active against all microbes. Antimicrobials used to treat or prevent infections are usually more specific e.g. antibiotics are active against bacteria, and antifungals are active against fungi. Even here there is variation. Some bacteria are naturally resistant against certain types of drugs, for example, and others are able to become resistant e.g. through genetic mutations. In today’s practical you will test the ability of different chemical compounds to inhibit the growth of specific microorganisms. Each pair of students will test the compounds against one species, and next week we will compare results from the whole class. First, you will inoculate agar plates to produce an even lawn of microbial growth. Then you will introduce chemicals into specific