2696

Microorganisms / General Information

extract DNA by using appropriate commercial DNA extraction kit.
2.4. Purification of PCR products
Various methods are available for removing unreachedsubstances (dNTP, prime, etc.). Purify according to the protocol of the method adopted.
2.5. Quantification of purified DNA
When purified DNA is measured by spectrophotometer,
calculate 1 OD260 nm as 50 mg/mL.
2.6. Labeling of PCR products with sequencing reagents
Use an appropriate fluorescence-labeled sequencing reagent suitable for the available DNA sequencer or its programand label the PCR products according to the instructions
provided with the reagent.
2.7. Purification of sequencing reagent-labeled PCR products
Transfer the product in 75 mL of diluted ethanol (7 in 10)
into a 1.5 mL centrifuge tube, keep in an ice bath for 20minutes, and centrifuge at 15,000 rpm for 20 minutes. Afterremoval of supernatant, add 250 mL of diluted ethanol (7 in10) to the precipitate and centrifuge at 15,000 rpm for 5minutes. Remove the supernatant and dry the precipitate.
2.8. DNA homology analysis
Place sequencing reagent-labeled PCR products in theDNA sequencer and read the nucleotide sequences of thePCR products. Compare the partial nucleotide sequencewith those in the BLAST database.
3.

Judgment
If sequencing data show over 90z identity with a sequence in the database, in general, judgment may be made asfollows.
( i ) In the case of bacteria, compare the nucleotides in
the product obtained with the 10F primer (the 800Fprimer when 800F/1500R primers are used) with theBLAST database. Higher ranked species are judged
as identified species or closely related species.
(ii) In the case of fungi, compare sequencing data for theproduct obtained with the ITS1F primer with theBLAST database. Higher ranked species are judged
as identified species or closely related species.
4. Reagents, Test Solutions(i) 0.5 mol/L Disodium dihydrogen ethylenediamine tetraacetate TS: Dissolve 18.6 g of disodium dihydrogen ethylenediamine tetraacetate dihydrate in water to make 100 mL.
(ii) 1 mol/L Tris buffer solution (pH 8.0): Dissolve 24.2 gof 2-amino-2-hydroxymethyl-1,3-propanediol in a suitableamount of water, adjust the pH to 8.0 with 0.2 mol/L hydrochloric acid TS, and add water to make 200 mL.
(iii) TE buffer solution: Mix 1.0 mL of 1 mol/L tris buffersolution (pH 8.0) and 0.2 mL of 0.5 mol/L disodium dihydrogen ethylenediamine tetraacetate TS, and add water tomake 100 mL.
(iv) DNA releasing solution: Divide TE buffer solution
containing 1 volz of polyoxyethylene (10) octylphenyl ether
into small amounts and store frozen until use.
(v) PCR reaction solution
10-fold buffer solution*
5 mLdNTP mixture**
4 mL
10 mmol/L Sense primer
1 mL
1 mL
10 mmol/L Anti-sense primer
Heat-resistant DNA polymerase (1 U/mL)
1 mL
Water
36 mL
* Being composed of 100 mmol/L 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (pH 8.4), 500 mmol/
L potassium chloride, 20 mmol/L magnesium chloride and0.1 g/L gelatin.

JP XVIII** A solution containing 2.5 mmol/L each of dGTP (sodium 2?-deoxyguanosine 5?-triphosphate), dATP (sodium 2?deoxyadenosine 5?-triphosphate), dCTP (sodium 2?-deoxycytidine 5?-triphosphate) and dTTP (sodium 2?-deoxythymidine 5?-triphosphate). Adequate products containing thesecomponents as described above may be used.
(vi) Sequencing reagent: There are many kinds of sequencing methods, such as the dye-primer method for labeling ofprimer, the dye-terminator method for labeling of dNTP terminator and so on. Use an appropriate sequencing reagent
kit for the apparatus and program to be used.
(vii) 50-Fold concentrated TAE buffer solution: Dissolve
242 g of 2-amino-2-hydroxymethyl-1,3-propanediol in 57.1
mL of acetic acid (100) and 100 mL of 0.5 mol/L disodium
dihydrogen ethylenediamine tetraacetate TS, and add water
to make 1000 mL.
(viii) 1-Fold concentrated TAE buffer solution: Diluted 50fold concentrated TAE buffer solution (1 in 50) prepared before use is referred to as 1-fold concentrated TAE buffer solution.
(ix) Agarose gel: Mix 1.5 g of agarose, 2.0 mL of 50-foldconcentrated TAE buffer solution, 10 mL of a solution ofethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) (1 in 100) and 100 mL of water. Afterdissolving the materials by heating, cool the solution toabout 609C, and prepare gels.
(x) Loading buffer solution (6-fold concentrated): Dissolve
0.25 g of bromophenol blue, 0.25 g of xylene cyanol FF and1.63 g of disodium dihydrogen ethylenediamine tetraacetate
dihydrate in 50 mL of water, and add 30 mL of glycerol andwater to make 100 mL.
(xi) PCR primers
For

PrimerBacteria

10F
800R
800F
1500R

5?-GTTTGATCCTGGCTCA-3?
5?-TACCAGGGTATCTAATCC-3?
5?-GGATTAGATACCCTGGTA-3?
5?-TACCTTGTTACGACTT-3?

Fungi

ITS1F
ITS1R5?-GTAACAAGGT(T/C)TCCGT-3?
5?-CGTTCTTCATCGATG-3?

(xii) Polyoxyethylene(10)octylphenyl ether: A pale yellow,
viscous liquid.

Rapid Counting of Microbes using
Fluorescent Staining <G4-8-152>
This chapter provides rapid methods using fluorescencestaining for the quantitative estimation of viable microorganisms. Incubation on an agar medium has been widelyused for quantitative estimation of viable microorganisms,
but a number of environmental microorganisms of interest
are not easy to grow in culture under usual conditions, thus
new microbial detection methods based on fluorescence orluminescence have been developed. In the fluorescence staining method, microorganisms are stained with fluorescentdye, and can easily be detected and counted with various
sorts of apparatus, such as a fluorescence microscope or
flow cytometer. Methods are available to detect total
microorganisms, including both dead and viable cells, or todetect only cells with a specified bioactivity by choosing thedye reagent appropriately. Nucleic acid staining reagents,
which bind with DNA or RNA, detect all cells containing

JP XVIIInucleic acids, whether they are live or dead. This technique isthe most fundamental for the fluorescence staining method.
On the other hand, fluorescent vital staining methods target
the respiratory activity of the microorganism and the activityof esterase, which is present universally in microorganisms.
In the microcolony method, microcolonies in the early stageof colony formation are counted. The CFDA-DAPI double
staining method and the microcolony method are described
below. These methods can give higher counts than the othertechniques, because these rapid and accurate techniques provide quantitative estimation of viable microorganisms basedon a very specific definition of viability, which may bedifferent from that implicit in other methods. The procedures of these methods described here may be changed as experience with the methods is accumulated. Therefore, otherreagents, instruments and apparatus than those describedhere may also be used if there is a valid reason for so doing.
1.

CFDA-DAPI double staining method
Fluorescein diacetate (FDA) reagents are generally used
for the detection of microorganisms possessing esteraseactivity. These reagents are hydrolyzed by intracellularesterase, and the hydrolyzed dye exhibits green fluorescence
under blue excitation light (about 490 nm). Modified FDAs
such as carboxyfluorescein diacetate (CFDA) are used because of the low stainability of gramnegative bacteria with
FDA. The principle of the CFDA-DAPI double staining
method, which also employs a nucleic acid staining reagent,
4?,6-diamidino-2-phenylindole (DAPI), is as follows. The
nonpolar CFDA penetrates into cells and is hydrolyzed tofluorescent carboxyfluorescein by intracellular esterase. The
carboxyfluorescein is accumulated in the living cells due toits polarity, and therefore green fluorescence due to carboxyfluorescein occurs when cells possessing esterase activity are
illuminated with blue excitation light. No fluorescent carboxyfluorescein is produced with dead cells, since they areunable to hydrolyze CFDA. On the other hand, DAPI binds
preferentially to the adenine and thymine of DNA after
penetration into both viable and dead microorganisms, andconsequently all of the organisms containing DNA exhibit
blue fluorescence under ultraviolet excitation light. Therefore, this double staining method enables to count specifically only live microorganisms possessing esterase activity
under blue excitation light, and also to determine the total
microbial count (viable and dead microorganisms) under ultraviolet excitation light.
1.1. Apparatus
1.1.1. Fluorescence microscope or fluorescence observation apparatus
Various types of apparatus for counting fluorescencestained microorganisms are available. Appropriate filters
are provided, depending on the fluorescent dye reagentsused. A fluorescence microscope, laser microscope, flow
cytometer, and various other types of apparatus may be used
for fluorescence observation.
1.2. Instruments(i) Filtering equipment (funnels, suction flasks, suctionpumps)(ii) Membrane filters made of polycarbonate (poresize:0.2 mm); A suitable filter that can trap particles on the surface can be used other than polycarbonate filter.
(iii) Glass slide(iv) Cover glass(v) Ocular micrometer for counting (with 10 × 10 grids)
1.3. Procedure
An example of the procedure using a fluorescence microscope is described below.

General Information / Microorganisms

2697

1.3.1. Preparation of samples
Prepare samples by ensuring that microbes are dispersed
evenly in the liquid (water or buffer solution).
1.3.2. Filtration
Set a membrane filter made of polycarbonate (poresize:0.2 mm) on the funnel of the filtering equipment. Filter anappropriate amount of a sample to trap microbes in the sample on the filter.
1.3.3. Staining
Pour sufficient amount of buffer solution for CFDA
staining, mixed to provide final concentration of 150 mg/mLof CFDA and 1 mg/mL of DAPI, into the funnel of thefiltering equipment and allow staining in room temperaturefor 3 minutes, then filter the liquid by suction. Poursufficient amount of aseptic water in the funnel, filter bysuction, and remove excess fluorescent reagent left on thefilter. Thoroughly dry the filter.
1.3.4. Slide preparation
Put one drop of immersion oil for fluorescence microscope on the glass slide. Place the air dried filter over it, withthe filtering side on the top. Then put one drop of immersion
oil for fluorescence microscope on the surface of the filter,
place a cover glass to enclose the filter. Put another drop ofimmersion oil for fluorescence microscope on the cover glass
when using an oilimmersion objective lens.
1.3.5. Counting
Observe and count under a fluorescence microscope, with
1000 magnification. In case of CFDA-DAPI double staining
method, count the microorganisms (with esterase activity)
exhibiting green fluorescence under the blue excitation lightfirst to avoid color fading by the ultraviolet light, then countthe microorganisms (with DNA) exhibiting blue fluorescence
under the ultraviolet excitation light in the same microscopic
field. Count the organisms exhibiting fluorescence on morethan 20 randomly selected fields among 100 grids observed
through an ocular micrometer of the microscope, and calculate the total number of organisms using the following formula. The area of the microscopic field should be previously
determined with the ocular and objective micrometers. The
amount of the sample to be filtered must be adjusted so thatthe cell number per field is between 10 and 100. It might benecessary to reprepare the sample in certain instances. (Insuch case that the average count number is not more than 2organisms per field, or where more than 5 fields are foundwhich have no organism per field, it is assumed that themicroorganism count is below the detection limit.)
Number of microbes (cells/mL)＝s(average number of microbes per visual field)× (area of filtration)t/s(amount of samplefiltered) × (area of one microscopic field)t
1.4. Reagents and test solutions(i) Aseptic water: Filter water through a membrane filter
with 0.2 mm pore size, then sterilize it by heating in an autoclave at 1219C for 15 minutes. Water for injection may beused.
(ii) CFDA solution, 10 mg/mL: Dissolve 50 mg of
CFDA in dimethylsulfoxide to prepare a 5 mL solution.
Store at －209C in light shielded condition.
(iii) Buffer solution for CFDA staining: Dissolve 5 g ofsodium chloride with 0.5 mL of 0.1 mol/L disodium dihydrogen ethylenediamine tetraacetate TS and diluted disodium hydrogen phosphate TS (1 in 3) to prepare 100 mL of solution. Add sodium dihydrogen phosphate dihydrate solution (1 in 64) to adjust the pH level to 8.5. Filter the solutionthrough a membrane filter with a pore size of 0.2 mm.
(iv) DAPI solution, 10 mg/mL: Dissolve 10 mg of DAPI