1 BIO335F Indoor Mould Experiments: Flowchart for Sampling Plan: November 2011 Hypotheses – Remember that the objective is to test the NULL – in order to prove the hypothesis, the null must be rejected: H1: There is a significant difference in fungal diversity and occurrence [the number of colony forming units (CFUs)] in daytime rest areas between cats and dogs. H1 NULL: There is no difference in fungal diversity and occurrence [the number of colony forming units (CFUs)] in daytime rest areas between cats and dogs. Treatments (4 pet situations to be compared): 1) 2) 3) 4) 5) Indoor-outdoor dog (DOG1) Indoor-outdoor dog (DOG2) Indoor cat only (ICAT Outdoor cat only (OCAT) No pets (NOPET) Sampling localities: one room from each locality where pet spends day (for no pet treatment use TV room): 1) DOG1: Prof. Kohn’s downstairs family room. 2) DOG2: Vincent Santiago 3) ICAT: Crystal Wolfram. 4) OCAT: Andrew Martinez-Novoa 5) NOPET: Olga Tkachenko Overall Sampling strategy: Air will be sampled by exposing without the lid for 3 hours, 3 petri dishes containing nutrient medium at two heights in the room: on the floor and on a table top for a total of 6 plates for each of the five localities (each house/apartment). Swab samples will be taken from two places in the room: (1) one taken from uncarpeted floor and (2) one from your choice, such as litter box or rim of water bowl for a total of 2 swabs from each of the four localities (each house/apartment). Dust vacuumed into a new, clean vacuum bag from both the floor and from the pet bed of each locality (each house/apartment): fold and tape full vacuum bag closed = 5 total. Please photograph the general state of each room sampled to allow us to compare the way each room looks. 1. AIR: Treatments: 1) Indoor-outdoor dog (DOG1) 2) DOG2: Vincent Santiago 3) Indoor cat only (ICAT) 4) Outdoor cat only (OCAT) 5) No pets (NOPET) Medium: Potato dextrose agar (PDA) and antibiotics (with 0.05 g/L Chloramphenicol & 0.05g/L % Tetracycline hydrochloride) Six plates for each of 5 localities/treatments Total plates for class: 30 Sampling technique: (1) LABEL your plates, eg. 07/11/11Dog1-AIR-Floor plate 1, (2) 07/11/11Dog1-AIR-Floor plate 2, (3) 07/11/11Dog1-AIR-Floor plate 3, (4) 07/11/11Dog1-AIR-table plate 1 –etc. Note use ICAT, OCAT, NOPET for the other three locality/treatments. 2 (2) How to sample: You are exposing 3 plates on the floor, and 3 plats on a table. Remove lid of petri dish, place with agar medium on upper side, facing up, for 3 hours. After 3 hours, replace lid and parafilm. Store upside down. (3) Incubate in Indoor Mould Experiment cupboard for 1 week. 2. SWAB: Treatments: 1) Indoor-outdoor dog (DOG1) 2) Indoor-outdoor dog DOG2 3) Indoor cat only (ICAT) 4) Outdoor cat only (OCAT) 5) No pets (NOPET) Collection medium: Buffer Number of swabs per room = 2. Number of swabs for each treatment/locality = 2 (one room for each treatment/locality). Total buffer tubes for class: 10 (30mL buffer/tube) = 300 mL total buffer. Sampling technique: (1) LABEL YOUR tubes: e.g. Eg. 07/11/11Dog1-SWAB-Floor, or 07/11/11Dog1-SWAB-Litter or 07/11/11Dog1-SWAB-BowlRim (2)Wearing your latex lab gloves, use one sterile Q-Tip to swab in 10 strokes each of your of your 2 areas (uncarpeted floor OR litter box rim/ water bowl rim), then put each used Q-tip in its own tube of buffer. Back in our UTM BIO 335 lab: FIRST, label your plates! Eg. 07/11/11Dog1-SWAB-Floor-PlateA, 07/11/11Dog1-SWAB-Floor-PlateB 07/11/11Dog1-SWAB-Litter-PlateA or 07/11/11Dog1-SWAB-BowlRim-PlateA 07/11/11Dog1-SWAB-Litter-Plate B or 07/11/11Dog1-SWAB-BowlRim-PlateB – note that the Plate B is either litter box or water bowl rim! Not both! Medium: Malt extract agar (MEA) with antibiotics (0.05 g/L Chloramphenicol & 0.05g/L Tetracycline hydrochloride). Add antibiotics to MEA (MEA must be lukewarm and ready to pour). Pour MEA + antibiotics into Petri dish. Vortex swab buffer and immediately swirl 100 µL of buffer into MEA + antibiotics. Place lid on Petri dish and allow to cool. Parafilm and Incubate in Indoor Mould Experiment cupboard for 1 week. Number of dilutions plated for each buffer tube sample: 1 Number of plates per dilution: 2 Total plates for class: 2 samples (floor or box/bowl) x 2 plates x 1 dilution x 5 treatment/localities = Total of 20 plates 3 3. DUST: Treatments: 1) 2) 3) 4) 5) Indoor-outdoor dog (DOG1) Indoor-outdoor dog (DOG2) Indoor cat only (ICAT) Outdoor cat only (OCAT) No pets (NOPET) Total vacuum bags for class: 5 (1 per treatment locality) Total number of sterile spatulas: 5 Total number of sterile plastic storage bags: 5 Pens and tape for labeling Indoor sampling technique: Vacuum floor and surface of pet bed, using a new, clean bag for each treatment/locality (total of four treatment/localities). Note! We need dust even if room is very clean – rugs are good, get under bed. Each time the vacuum is used, it will be cleaned and dried thoroughly by Susan Dixon or under her direction – before it is used by the next person for a new locality. Back in our UTM BIO 335 lab: FIRST, label your TUBES! For Example: Dog1- .10 (USE 10 ml tube) Dog1- .01 (USE 9 ml tube) Dog1- .001 (USE 9 ml tube) Dog1- .0001 (USE 9 ml tube) SECOND, label your PLATES! For Example: 07/11/11Dog1-DUST07/11/11Dog1-DUST07/11/11Dog1-DUST07/11/11Dog1-DUST- .001-Plate A .001-Plate B .0001-Plate A .0001-Plate B 07/11/11Dog2DUST07/11/11Dog2-DUST07/11/11Dog2-DUST07/11/11Dog2-DUST- .001-Plate A .001-Plate B .0001-Plate A .0001-Plate B Then continue for: 07/11/11-ICAT-DUST 07/11/11-OCAT-DUST 07/11/11-NOPET -DUST Media: We are using one kind of medium, Rose Bengal Agar For each of the four dust samples, weigh out 1 gram of dust and place in tube of 10 ml peptone broth (this is a 0.10 dilution). VORTEX to blend! Make 0.01, 0.001, and 0.0001 dilutions using each tube, i.e. vortex the 0.10 dilution and transfer 1 ml of the dilution to 9 ml broth. This is your 0.01 dilution. Transfer 1 ml of the 0.01 dilution to 9 ml broth for your 0.01 dilution, etc. Always vortex previous dilution before transferring to broth. The dust settles quickly. Take 1 ml of your final 2 dilutions (0.001 and 0.0001), and swirl into RBA. *Remember to vortex dilution immediately prior to pouring in medium. Place lid on Petri dish and allow to cool. Parafilm and incubate for 1 week in the BIO 335 culture cabinet . Total number of sampled treatments/localities = 5. 4 Total tubes of peptone broth for dilution series : 20 – This includes 5 tubes with 10mL peptone broth/tube, and 15 tubes with 9mL peptone broth/tube = 185 mL total peptone broth. Total number of dilutions plated: 2 Total number of plates per dilution: 2 Total RBA for class: 2 x 2 x 5 = 20 plates Formulas for media and buffers used in 2011 Indoor Mould Experiment Buffer for Swabs: 0.0425g KH2PO4 0.25g MgSO4 0.008g NaOH 0.2 ml Tween 80 pH 7.0 ∀ 0.1 1000ml Distilled water Mix above ingredients, autoclave. Dispense 30 ml asceptically into sterile 50 ml Falcon tubes. Antibiotic Stock Solution: 0.5g Chloramphenicol DISOLVE in ETOH first (10 ml0 0.5g Tetracycline hydrochloride 40 ml double distilled H20 Mix together and filter sterilize into sterile tube. Refrigerate. Use 5 ml of stock/L of medium to get final concentration of 0.05g/L Streptomycin stock Solution: 1g streptomycin sulphate in 100 ml sterile distilled water. Filter sterilize and add 8ml of solution to 1000ml of media asceptically after media has been autoclaved and before pouring into plates. Rose Bengal Stock solution: 1 gm Rose bengal in 300ml distilled water. Add at a rate of 1 ml stock to 100 ml media (add 10ml to 1 litre of media). Should be added to media after other ingredients have been dissolved but before autoclaving.