Manual for the PhenePlate system ver. 2.3 08-11-02

MANUAL FOR THE PHENE-PLATE SYSTEM

Contents

A. General information 2

B. Products 2

B.1 High resolution PhP plates 2

B.2 Rapid screening PhP plates 2

B.3 PhP suspending medium 2

C. Test procedure 3

C.1 Materials and equipment required 3

Materials 3

Equipment 3

C.2 Preparation of PhP suspending medium 3

Bromothymol blue stock solution (0.11%) 3

Compositions of suspending medium for various bacteria 4

C.3 Fingerprinting procedure 5

C.3.1 Pre-cultivation of bacteria 5

C3.2 Setting up an assay 5

C4. Preparation of bacterial suspensions (PhP-48 plates, or 24-test plates when one medium vial per bacterial isolate is used) 5

C5. Inoculation of PhP-plates 5

C5.2. After inoculation 6

C6. Handling of Rapid Screening PhP plates and rapid handling of 24-test plates 6

C7. Incubation of PhP-plates 7

C8. Reading of test results 7

C8.1 Readings with a microplate reader 7

C8.2 Visual reading of tests 7

C8.3 Reading with a fladbed scanner 7

See manual for PhPWIN software 7

D. Some hints 8

D.1. Reproducibility 8

D2. Inoculum density 9

D.3. Some other hints 9

D.4. Control experiments 9

E. Analysis of results 10

F. Reagent lists 11

Reagents in the PhP-48 general plate 11

Reagents in the PhP-EC plate for E.coli 12

Reagents in the PhP-FS plate for Fecal streptococci 12

Reagents in the PhP-CS plate for coagulase-negative staphylococci 12

Reagents in the PhP-Rapid screening plates 13

G. Control plate – Comparison between different reading methods 14

For problems and comments, please contact by E-mail.

You can also look at www.phplate.se for most recent update of the manual

A. General information

The PhenePlateTM system is an automated system for simple and rapid subtyping of bacteria for epidemiological, nosocomial and ecological studies. The system is based on the evaluation of the kinetics of biochemical reactions (by reading test results at several occasions), performed in 96 well microplates. The microplates (PhP plates) contain 2 to 8 sets of dehydrated reagents, which have been specifically selected for various groups of microorganisms.

B. Products

B.1 High resolution PhP plates

High resolution plates differentiate each isolate on 24 or 48 different reagents. The high resolution plates are used when a careful typing of each isolate is needed (e.g. for epidemiolgical investigations). The PhP-48 plates are also used for bacteria for which no special plates are available

PhP-plate / Isolates per plate / Reagents per assay / Bacteria
24-test plates
PhP-EC / 4 / 24 / E.coli
PhP-CS / 4 / 24 / Staphylococci
PhP-FS / 4 / 24 / Enterococci
48-test plates
PhP-48 / 4 / 48 / All metabolically active bacteria

B.2 Rapid screening PhP plates

Rapid screening PhP plates differentiate each isolate on 11 reagents. Rapid screening plates are particular suitable for ecological studies involving large numbers of isolates, when the information of the whole population is more important than the information on each individual isolate

PhP-plate / Isolates per plate / Reagents per assay / Bacteria
PhP-RE / 8 / 11 / E.coli
PhP-RS / 8 / 11 / Enterobacteria (Salmonella, Klebsiella, Enterobacter, etc)
PhP-RF / 8 / 11 / Enterococci
PhP-AE / 8 / 11 / Aeromonas spp
PhP-RV / 8 / 11 / Vibrio spp
Storage and shelf life

Packages of PhP plates that are kept at –20oC can be stored for several years. If they are stored refrigerated (+4oC) in unopened packages, they can then be kept for at least 6 months after purchasing date. They may also be stored for shorter times (at least 2 weeks) in room temperature. Open packages should always be stored frozen or refrigerated and should be used within one month.

B.3 PhP suspending medium

PhP suspending medium is available in flasks with 200 ml (for 10 PhP plates). The medium can be kept at room temperature for at least three months, and in a refrigerator for at least one year.

The suspending medium can easily be made in your own laboratory (see below).

C. Test procedure

Note! The bacterial phenotypes obtained by the PhP system are based on the results from comparisons between the assayed isolates, and not on the test results obtained. Therefore the reaction conditions (pre-cultivation, suspending media compositions, incubation temperature, incubation time etc.) may be freely selected for a particular investigation by the user, and thus the procedures described in this manual may be altered. However, it is important that all isolates that are to be compared to each others are always treated in the same way.

Note! Also read section D of this manual if you are not familiar with the assay

C.1 Materials and equipment required

Materials

·  PhP-plates (microplates with dehydrated reagents)

·  Lids for microplates (can be soaked in alcohol and reused) (lids are optional - also plates with clean bottoms can be used as covers)

·  PhP suspending medium. (The PhP suspending medium can be easily prepared in the laboratory, see C2 below). If you have purchased PhP suspending medium and are using PhP-48 plates (or PhP 24 test plates, optional – see below) you also need sterile flasks or test tubes for preparation of 12 (or 8) ml suspension of each bacteria to be tested.

·  Sterile pipette tips (can be rinsed, autoclaved and reused).

·  Sterile paraffin oil

·  Inoculating loops (or autoclaved tooth sticks), sterile petri dishes

Equipment

·  Steppable 1.5 ml multichannel pipette (e.g. multistepper from Labsystems)

·  For PhP rapid screening plates preferably also a steppable 300 ml multichannel pipette

·  Containers for inoculated plates (wet chamber – e.g. plastic boxes with lids).

·  Incubator

·  PC-compatible computer with PhP software.

·  Spectrophotometer for micro plates (or light table for visual readings of plates). Instead of a spectrophotometer, a scanning device (that can scan transmissible originals) connected to a computer, and a software that can copy images to the clipboard or can generate BMP or JPG files can also be used.

C.2 Preparation of PhP suspending medium

Bromothymol blue stock solution (0.11%)

1.1 gram of bromothymol blue is dissolved into 90 ml distilled water. Add 10 ml 1M NaOH. Stir until all indicator has dissolved (check carefully), and add 900 ml of distilled water. If the solution is to be stored it should be autoclaved. This solution is enough for 10 liter of PhP suspending medium

Note! It is very important that the indicator solution has the right concentration, and different brands of bromothymol blue may vary in strength. Check the stock solution by diluting 1 ml with 8 ml of water and 1 ml Phosphate buffer (c:a 0.2M) at pH 7.5. Add 0.150 ml of this solution to some of the wells of a micro plate, and measure the absorbances at 620 nm. The absorbance values of the wells with this solution should be between 1.9 and 2.3. If lower, add some more bromothymol blue, and if higher, add some more water

Compositions of suspending medium for various bacteria

The normal suspending mediums for various bacteria are given below . These recommended compositions of mediums may well be altered. However, always use the same medium for all bacteria in a test series.

If fastidious microorganisms are to be investigated, suspending mediums which corresponds to the requirement of these microorganisms must be prepared. However, these mediums should not contain any fermentable carbon source.

PhP plate/bacterial species
/ Nutrient source / Amount
g/l /
Other components
/ Amount
PhP-RE, PhP-RS, PhP-EC, PhP-48 / Proteose pepton / 1.0 / Phosphate buffert*
/ 8 ml/l*
PhP-48/Klebsiella / Proteose peptone / 0.5 / Phosphate buffert* / 8 ml/l*
PhP-FS, PhP-RF
PhP-CS / Proteose peptone
Yeast extract / 2.0
0.5 / Sodium chloride
Phosphate buffert* / 5.0 g/l
20 ml/l*
PhP-AE / Proteose peptone / 1.0 / Phosphate buffert* / 8 ml/l*
PhP-RV / Proteose peptone / 0.5 / Sodium chloride
Phosphate buffert* / 20 g/l
8 ml/l*

*Indicates a 0.2 M solution of phosphate buffert (Na2HPO4 + NaH2PO4) at pH 7.5

For making up 1 liter of medium, use the following procedure:

Add the above nutrient sources and the other components when appropriate (Sodium chloride and phosphate buffert) to 900 ml distilled water. Stir until everything has dissolved. Add 100 ml of Bromo Thymole blue stock solution (see above). Adjust pH to 7.8- 8.0 with diluted hydrochloric acid or sodium hydroxide.

For the high resolution PhP-48 plates the solution is divided in portions in capped vials, one vial per isolate to be assayed. The volume of medium in each flask should be 12-15 ml

For other high resolution PhP plates (24-test plates) the solution is divided in portions in capped vials, one vial per isolate to be assayed. The volume of medium in each vial should be 8 - 10 ml. An alternative and more rapid way is to inoculate these plates in the same way as the rapid screening plates (see below), and in that case the medium is divided into larger volumes (100-500 ml, 20 - 25 ml per plate will be required).

For rapid screening PhP plates (12-test plates) the medium is divided into larger volumes (100-500 ml per flask).

The vials or flasks are autoclaved (not higher than 120oC) for 15 minutes and stored in a refrigerator until they are used. Normal media with only peptone can be stored for at least one year. The medium can also be stored at room temperature (below 25oC) for at least 3 months.

Note!! Measure the color of the medium before it is used the first time. Add 0.150 ml of the medium to the wells of an empty microplate. Measure the absorbance at 620 nm. The absorbance values should be between 1.9 and 2.2. If not, the indicator concentration might have been to low, or the pH of the medium might be wrong.
If the absorbance values have decreased during autoclavation, the medium might have been too hard autoclaved

C.3 Fingerprinting procedure

Note! Since the PhP system is based on bacterial metabolism of various substrates, it is necessary to avoid contamination by other bacteria. Always use sterile materials, and use sterile techniques throughout the fingerprinting procedure. Avoid splashes and spills between wells.

C.3.1 Pre-cultivation of bacteria

The bacteria to be tested are first pre-cultivated on an appropriate agar medium, e.g. nutrient agar. Use the same pre-cultivation conditions for all strains in a test series, and avoid using agar cultures older than 1 week for inoculation of the PhP plates. If bacteria from e.g. membrane filtered water samples are tested on rapid screening plates, the bacterial colonies may be picked directly from the original isolation plate (see C6 below).

C3.2 Setting up an assay

Select one or a few bacterial strains that are to be used as references and include these every time an assay is performed (IMPORTANT!). If you are not familiar with the system, test these isolates in duplicates, in order to establish your own intra-assay reproducibility. When using the rapid screening PhP plates, always include a negative control (i.e. only medium) in the last row of the last plate

C4. Preparation of bacterial suspensions (PhP-48 plates, or 24-test plates when one medium vial per bacterial isolate is used)

(For 12-test PhP plates or for 24-test plates when the substrate is divided into larger volumes see C6 below)
If you have purchased PhP suspending medium in 200 ml bottles, first dispense medium into sterile capped vials, 8 ml per vial for 24-test plates, and 12 ml per vial for 48-test plates.
Use a sterile inoculation loop to pick bacteria from the agar plates and suspend into the vials containing the suspending medium. The system is not very sensitive to bacterial concentration, thus the amount of bacteria may vary, but should preferably be between 0.3 and 3 mg (wet weight) ("the edge of a loop", or 1 to 10 colonies, depending on the size of the colonies). Leave the suspensions for at least 30 minutes. In order to obtain a homogeneous bacterial suspension, shake the vials vigorously before the inoculation
Hint. As last sample in an assay, use a negative control (i.e. only medium) /

C5. Inoculation of PhP-plates

Use a multichannel pipette with sterile tips to take the suspension directly from the vial (remember not to draw the bacterial suspension to high - this will result in contamination of the suspension), or first pour the suspension into a sterile petri dish. Inoculate 0.150 ml of the bacterial suspension into each of the wells of the PhP-plates in the following way: /
For 24-test PhP plates:
Isolate 1 in rows A-B
Isolate 2 in rows C-D
Isolate 3 in rows E-F
Isolate 4 in rows G-H
For PhP-48 plates:
Isolate 1 in rows A - D
Isolate 2 in rows E – H

Hint: In order to save disposable materials, pipette tips used for inoculation may be rinsed, autoclaved and reused. Lids may be soaked in alcohol, dried in clean paper bags, and reused.

C5.2. After inoculation

The inoculated plates are covered with sterile lids, or with another microplate. They are then kept in a refrigerator until they are put into the incubator.

NOTE ! The plates must be handled very carefully after the inoculation. Splashes or spills might result in contamination between wells.

Proceed to C7 below!

C6. Handling of Rapid Screening PhP plates and rapid handling of 24-test plates

First, use a multichannel pipette with sterile tips to fill all wells in the PhP-plate with suspending medium.
For 12-test plates, dispense 0.320 - 0.375 ml into all eight wells of column 1 (the inoculation wells) in the plate, and 0.150 ml into all other wells. Proceed with the other plates in the assay
For 24-test plates, first dispense 0.150 ml into all wells of all plates in the assay. Remove every second tip from the multichannel pipette and dispense 0.220 - 0.275 more medium into rows 2, 4, 6, and 8 of column 12 in the plates (the inoculation wells) . /
Use a sterile tooth stick (or inoculation loop) to pick a single bacterial colony from the agar plate and suspend it into the first inoculation well (column 1 of row A for 12-test plates, column 12 of row B for 24-test plates). With another inoculation loop pick another bacterial colony and suspend it into the next inoculation well (column 1 of row B for 12-test plates, column 12 of row D for 24-test plates). Proceed until all 8 (or 4) inoculation wells have been inoculated with different bacteria. Proceed with the other plates in the assay. The amount of bacteria should be between 0.1 and 1 mg (wet weight) ("the edge of a loop").
The plates are left for at least 1 hour, hereafter the bacterial suspensions in the inoculation wells are homogenized by sucking up and blowing down a couple of times by the aid of a multichannel pipette with 8 (for 12-test plates) or 4 (for 24-test plates) tips (careful - the suspension must not touch the pipette).
0.010 - 0.025ml (for 12-test plates) or 0.010-0.0125ml (for 24-test plates) of the bacterial suspensions in the inoculation wells are then transferred to all other 11 or 23 wells in each one or two rows by the aid of the multi-channel pipette. Cover the plates with sterile lids or another PhP plate, and, to avoid drying, put them into a wet chamber. /

Note!! The PhP rapid screening plates contain only 11 reagents, and thus small variations in only one test, due to e.g. small variations in assay conditions, might influence the reproducibility. It is therefore advisable to run all isolates to be compared in the same assay. Since up to 500 isolates may easily be run during the same day, this should be no problem. If isolates need to be compared from different assay occasions, make sure to use several reference strains (at least 3-4), and run these in all assays.