MICROBIAL COLONY REPLICATOR User manual
Contents
1. 6 Check alignment by making an ink print as above 9 Flooding the HGMF Page 5 of 6 These Figures excellently illustrate the importance of controlling pressure time and also avoiding having a concave agar surface when replicating HGMF 1 A master HGMF prepared from diluted bovine faeces on Violet Red Bile agar HGME 2 This replicate of HGMF 1 on Oxoid Chromogenic E Coli agar clearly differentiates species HGMEF 3 A further replicate on Mueller Hinton antibiotic agar has reproduced the centre of HGMF 1 correctly but there is a ring of positive grid cells around the outside The agar may have been concave due to incorrect drying greater contact pressure time on the HGME at the edge of the Inoculator compared with its centre increased the risk of flooding Photos courtesy of Dr D Jordan New South Wales Agriculture Wollongbar NSW Australia Page 6 of 62. Heightsfocus Clamp Knob Ti Operating Lever i EF F i i Crank Follower up A cami Lewer Boss Shaft Dock Adjust Boss I Crank Cower _ Dock Clamp Knobs lat Plate Inoculator Locking Lewer i Check Ring Inoculator Dock i Locators F Fost HG iF focus plane HGMF Slider w Erg Front amp side views of the Microbial Colony Replicator Inoculator locked into the Inoculator Dock Locking Lever in locked position Page 2 of 6 2 Replicating keep in mind what s happening Remember that i An HGMF is very flexible each Inoculator points deforms it and presses it into the agar ii The agar is like a sponge the instant it is pressed it expresses water which floods back up into the HGMF grid cell If pressed for more than a second or so water will swamp and contaminate adjacent grid cells Agars for different purposes have different sponginess The skill to learn in replicating is to bring the Inoculator down onto the HGMF just firmly enough and for long enough that floodwater washes some microbial cells from the Inoculator point But be careful not to press for too long or you will flush microbes into adjacent HGAMF grid cells see 9 Flooding the HGMF One second or less contact is enough As agar plates dry the agar becomes harder and its surface becomes concave This can cause incomplete inoculation It is preferable to use plates containing 12 15 ml of fresh agar The ample agar lay
3. that a test print registers satisfactorily on an HGMF You will need a rubber stamp type ink pad and an HGMF or a photocopy Note that along one border HGMFs have a white T design that allows them to be registered under the Replicator crosshairs 1 Lock an Inoculator in the Inoculator Dock Ink the Inoculator by bringing it down onto the ink pad 3 Lay the HGMF on the HGME Slider centre its T under the microscope crosshairs and make a print If the plane of focus is too high use a pad under the HGMF 4 Check that ink dots in the print fall within their proper grid cells over the full area of the HGME Note it is not essential that dots be perfectly centered in all grid cells but the better the alignment the greater the tolerance you have when copying colonies In fact HGMFs vary slightly from one another In the event that the print is out of register use the Lineup Tool to re align the Replicator Lock the Lineup Tool in the Inoculator Dock Use the Operating Lever to drop the Lineup Tool s cross lines down into focus Slacken off both Dock Clamp Knobs Gripping both Dock Clamp Knobs between thumb and forefinger slide the Dock around until the Lineup Tool s cross lines register with the microscope crosshairs 5 Retighten the Dock Clamp Knobs Hint alternately partially tighten opposite sides until Dock is tight Do not over tighten POLS Replicator Lineup Tool matches the HGMF T
4. MICROBIAL COLONY REPLICATOR for ISO GRID HGME hydrophobic grid membrane filters User manual This Replicator permits preparation of HGMEFs that accurately copy the colony pattern of an HGMF master The ability to incubate replicates under a range of growth conditions is a useful diagnostic and research tool Replicating HGMFs is a simple three step process viz 1 Lock an Inoculator in the Replicator 2 Register the HGMF master under the microscope crosshairs and pull the Operating Lever to charge the Inoculator 3 Register a fresh HGMF under the crosshairs and pull the Lever to transfer a replica inoculum to it However there is a skill to learn and points to keep in mind Also this instrument differs in several ways from earlier Replicators Please take a few moments to read this Manual Contents 1 Replicator components 2 Replicating keep in mind what s happening 3 Replicating getting ready 4 Replicating charging an Inoculator from the master HGMF 5 Replicating making the copies 6 Cleaning sterilization and care of Inoculators 7 About Focus 8 Lining up the Replicator ISO GRID HGMF is a registered trademark of QA Lifesciences Inc San Diego CA USA FILTAFLEX Ltd POBox 1224 Almonte Ontario KOA 1A0 Canada Tel 613 256 3066 Fax 613 256 8681 www filtaflex ca E mail tsharpe filtaflex ca Page of 6 1 Replicator components Operating Lewer hiicroscope raised position eyepiece
5. er will ensure that the agar is resilient and planar enough to inoculate properly The agar surface should be planar but does not have to be uniformly thick The compressible HGME Slider allows the Replicator to accommodate plates gelled on a non horizontal surface 3 Replicating getting ready have sufficient sterilized Inoculators ready lay sufficient sterile HGMF on their respective agar plates check the Replicator alignment 4 Replicating charging an Inoculator from the master HGMF i Use the Operating Lever to raise the Inoculator Dock Move the Inoculator Locking Lever to the left and slide a sterile Inoculator into the Locators Touch only its wings ii Turn the Inoculator Locking Lever to the right to lock the Inoculator iii Withdraw the HGMF Slider place a master HGMF plate on it then slide it under the Inoculator Note If you try to slip a plate under an Inoculator without withdrawing the HGMF Slider you may touch and contaminate the Inoculator iv Align the HGME T bar under the microscope crosshairs Absolutely perfect registration is not essential experience will show how accurate you must be Keep fingertips in contact with both Base and HGMF Slider to steady them whilst aligning v Use the Operating Lever to bring down the Inoculator The weight of the Inoculator Dock and Shaft will usually provide sufficient force on the HGMF The force can be increased by pulling the Operating Lever gently do
6. h brush Inoculators between uses just sterilize them a slight build up of denatured organic material actually improves the ability of the points to hold inoculum Do not use replication for in situ PCR procedures even if DNAase treatments are employed since the point surfaces may protect DNA iv If it appears that some warping has occurred test prints made on a flat paper repeatedly miss in the same area dress Inoculators by rubbing them gently on medium grade e g 320 grit carborundum paper on a flat surface Do not rub off excessive amounts both point size and a tendency to spread microbes to adjacent HGMF grid cells will increase 7 About Focus The Replicator height was supplied adjusted so that the plane of an HGMF is in focus when it is on an agar plate of normal thickness on the HGMF Slider There is no rack and pinion if HGMFs on agar plates are not in focus loosen the Check Ring and Height Clamp knobs and raise or lower the whole body of the instrument Raising or lowering the instrument body will not affect alignment since all components move simultaneously 14 Lay Inoculator TEETH DOWN 2 Fold 3 Fold again twice on wrap 10 15 cm square 4 Invert and place on tray TEETH UP Wings flat on tray Do not stack Inoculators wrapped this way will be properly oriented when unwrapped Page 4 of 6 8 Lining up Although the Replicator will rarely go out of alignment it is advisable to check every week
7. not use excessive force vi A smooth down up movement without pause is usually sufficient More than 1 sec pause will usually swamp the HGMF vii Push the Operating Lever back to its rest position viii Make HGME copies without delay microbes die off quickly on a drying Inoculator Page 3 of 6 5 Replicating making the copies Make replicates by repeating this process on the fresh HGMFs The Inoculator will inoculate 100 of the grid cells of the first daughter HGMF Because it loses microbial cells to the fresh HGMF its ability to provide 100 inoculation decreases for succeeding replicates The number of replicates one can make before incomplete replications occur depends very much on the type of colonies on the HGMF master it may vary from five to 20 or more In general using a fresh Inoculator is preferable to recharging one that has been used 6 Cleaning sterilization and care of Inoculators i Ifused Inoculators are wrapped and autoclaved as shown they will be points down when unwrapped to avoid the chance of contaminating them by turning them over Alternatively autoclave them without wraps in covered trays ii Epoxy Inoculators withstand mild acids alkalis chlorine disinfectants and normal 121 C sterilization Avoid warping by autoclaving points up on a flat surface and do not stack them Do not soak aluminum Inoculators in alkaline solutions iii For many purposes it is not necessary to clean was
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