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 ve
ally 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
oken 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 co
ect 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
•
eath/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
ca
on 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
een 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
esults. 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,
ead) 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/ XXXXXXXXXXdrop 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
ench.
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
esistant 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