Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Summary

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Abstract

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

Introduction

Infertility is a condition that affects 10-15% of the human population of reproductive age ¹, from which nearly one-half seek medical treatment ². Although the etiology of infertility is diverse and in many cases multifactorial, the most common genetic abnormality in humans is embryonic aneuploidy ³. Aneuploidy is defined as the deviation (either gain or loss) of the correct number of chromosomes in a cell. The phenomenon of aneuploidy in human embryos is common and increases with advanced maternal age ^4,5. Four randomized controlled trials have highlighted the benefit of selecting only chromosomally normal (euploid) embryos for uterine transfer because this strategy resulted in increased implantation rates, lower miscarriage rates and a shorter time for achieving pregnancy ^6,7,8,9. Therefore, understanding the etiology of human aneuploidy can have important implications in assisted reproduction.

Although pre-implantation genetic testing for aneuploidies is beneficial in infertility treatments, a thorough understanding of how aneuploidies originate is still lacking. It is widely accepted that there is a positive correlation of meiotic aneuploidies (originated during gamete production) and maternal age, however, some women present embryonic aneuploidy rates that deviate from the mean rate for their given age ⁴. These cases suggest that age alone is not always predictive of the risk of generating an aneuploid embryo. Other factors may play a role in increasing the risk of embryonic aneuploidy, such as gene variants.

A key aspect of investigating the potential contribution of a gene variant to aneuploidy during oocyte meiosis is to design a system to rapidly evaluate meiotic gene function. Due to ethical constraints and limited access, it is impractical to perform these experiments using human eggs. These issues can be circumvented by using mouse oocytes, and here a series of assays to assess human gene function during meiosis I are described. By microinjecting the messenger RNA (mRNA) coding for the gene variant of interest, the localization of the human protein in the mouse egg can be visualized and used to determine if the ectopic expression of the wild-type and mutated human protein results in any phenotypic alterations that could lead to aneuploidy. These phenotypes include an increase in microtubules that attach to the improper to sister kinetochore and the inability to support chromosome alignment at metaphase of meiosis I. Importantly, this protocol can be used to investigate both gain and loss of function genetic variants by establishing specific experimental conditions to challenge key events in oocyte meiosis such as spindle building and chromosome alignment ¹⁰.

Protocol

1. Molecular Cloning

1. Obtain full-length coding sequence of the gene of interest and the plasmid in vitro transcription (pIVT) vector¹¹.
   NOTE: Full-length cDNA clones are commercially available from various vendors or can be generated via reverse transcription polymerase chain reaction (RT-PCR). The National Center for Biotechnology Information (NCBI) online resource provides transcript sequences for genes from the Nucleotide and Expressed sequence tagged (EST) databases. For ease of protein visualization and analysis of expression levels, fusion to green fluorescent protein (Gfp) or another suitable fluorescent tag may be desired in the cloning strategy.
2. Using a validated cloning strategy, insert the coding sequence of the gene of interest into the multiple cloning region of pIVT. A detailed description of molecular cloning and the required steps have previously been described ¹².
   NOTE: If a gene-fusion product is to be generated, be sure that the cloning is in the proper reading frame.
3. Sequence the construct using Globin primers to ensure correct gene insertion, proper in-frame fusion, and that no polymerase-induced point mutations were created.
   NOTE: If Sanger DNA sequencing equipment is not available, many companies offer low cost DNA sequencing options.
4. Mutagenize DNA if generating single nucleotide polymorphisms (SNPs) or insertions/deletions (INDELS). DNA mutagenesis is described in steps 1.5-1.7 below. For wild-type constructs, proceed with the linearization step 1.7.
5. Design mutagenesis primers and complete the mutagenesis PCR reaction to generate mutant construct per manufacturer's protocol. PCR-mediated site-directed mutagenesis has been previously described ¹³. Additionally, many companies offer site-directed mutagenesis kits which include protocols.
6. Transform mutagenized DNA into bacterial host. Isolate and purify plasmid.
   NOTE: Many companies make kits that can be used to easily isolate and purify plasmid DNA. Follow manufacturers protocol accordingly. If using a gram-negative bacterial strain such as E. coli, it is recommended to use a kit that removes endotoxins.
7. Sequence the isolated DNA from bacterial transformants using standard Sanger DNA sequencing to confirm introduction of the desired SNP or INDEL.
8. Linearize final products using single restriction enzyme digestion of the purified DNA.
   NOTE: The pIVT vector contains multiple single-enzyme cut sites listed in the plasmid map on Addgene ¹⁴.
9. Ensure that the product is linear via agarose gel electrophoresis. The methodology for agarose gel electrophoresis has previously been defined ¹⁴.
   NOTE: For all remaining steps, use barrier (filter) pipet tips and RNase-free materials to ensure production of stable, high quality RNA.
10. Purify the digested product using a validated DNA clean-up protocol or kit, and elute in 30 µL RNase/DNase-free water.
    NOTE: Silica-matrix spin column-based kits are recommended for high quality, purified DNA. Follow manufacturers protocol accordingly.
11. In vitro transcribe DNA using T7 polymerase following the manufacturer's protocol.
12. Purify and elute RNA in 30 µL of RNase/DNase free water using a silica-matrix spin column-based nucleic acid purification kit following the manufacturer's protocol.
13. Perform denaturing agarose gel electrophoresis using formaldehyde to confirm the final RNA product size. See steps 1.13-1.22 below ¹⁴. (Figure 1).
14. Prepare gel by heating 1 g agarose in 72 mL water until dissolved, then cool to 60 °C.
15. Prepare 10X MOPS running buffer by adding 0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, and 0.01 M EDTA.
16. Add 10 mL of 10X MOPS running buffer, and 18 mL of 37% formaldehyde (12.3 M) to the cooled agarose solution.
17. Caste the gel by pouring the agarose solution into a gel-forming container. Use a comb large enough to accommodate 10 µL. After gel has set and is firm to the touch, gently remove the comb.
18. Assemble the gel in the electrophoresis tank, and add enough 1X MOPS running buffer, ensuring the gel is completely covered.
19. Prepare RNA sample by adding 1 µg of RNA to 0.5X volumes of formaldehyde-containing loading dye.
20. Add ethidium bromide to RNA solution at a concentration of 10 µg/mL.
21. Denature RNA sample solution at 70 °C for 5 min.
22. Using sterile, 10 µL filter tips, load 5 µL of molecular weight marker and 5 µL of RNA sample(s) into separate wells of the gel and electrophorese at 5 V/cm until the dye has migrated at least two-thirds of the length of the gel.
23. Visualize the RNA in the gel on a UV trans illuminator. Ensure the RNA is the correct size by comparing to the molecular weight marker.
24. To measure the concentration and purity of the RNA, transfer 1.2 µL of purified RNA using sterile 10 µL filter tips onto a UV-spectrophotometer.
    Note that high quality RNA should have absorbance ratios of A260/280 (1.8-2.2) and 260/230 (>1.7) respectively.
25. Using 10 µL filter tips, aliquot the remaining purified RNA into sterile 0.5 µL centrifuge tubes (3-5 µL/tube). Store tubes at -80 °C until ready for microinjection.
    NOTE: A final injection concentration of 500 ng/µL is an optimal starting point. It is recommended to dilute RNA 1:2 in RNase free H₂0 prior to microinjection to reduce stickiness of RNA in microinjection pipette.

2. Kinetochore-Microtubule Attachment Assay

1. Divide oocytes into equal groups for microinjection.
   NOTE: Oocyte collection, transfer, microinjection, and meiotic maturation has been described in detail in JoVE previously ¹⁵.
2. Microinject one group with experimental mRNA and the other group(s) with the WT control and PBS or Gfp mRNA.
   NOTE: 500 ng/µL is the recommended injection concentration to begin with ¹⁰. A picoinjector can be used to control injection amounts. An injection volume of 5-10 pL is recommended ¹⁵.
3. Using a hand or mouth operated pipetting apparatus where the glass pipette opening is slightly larger than the diameter of an oocyte (100-150 µm), transfer oocytes into a 100 µL drop of milrinone-free culture medium in a Petri dish covered in embryo quality mineral oil and incubate oocytes for 7 h at 37 °C in 5% C0₂ to allow sufficient maturation to metaphase of meiosis I (Met I) ¹⁵.
4. After incubation, using the same pipette apparatus as above, transfer oocytes into a center-well organ culture dish containing 700 µL of pre-chilled minimum essential media (MEM) collection medium in the center well and 500 µL H₂0 in the outside ring and place dish on ice for 7 min.
   NOTE: It is recommended to pre-chill MEM media and center well organ culture dishes at -20 °C for at least 20 min prior to treatment of oocytes.
5. Fix the oocytes by transferring them using the pipette apparatus into a well of a clear glass spot plate containing 400 µL of 2% paraformaldehyde in 1X PBS for 20 min at room temperature.
6. Using same pipette apparatus, transfer the oocytes into another well on a spot plate containing 400 µL blocking solution (PBS + 0.3% BSA + 0.01% Tween-20 + 0.02% NaN₃) and store at 4 °C until ready to process for immunostaining.
   NOTE: For storing oocytes at 4 °C, cover glass spot plate with parafilm to prevent evaporation.
7. Using same pipette apparatus, transfer oocytes to a new well on a spot plate containing 400 µL permeabilization solution (PBS + 0.3% BSA + 0.1% TritonX-100 + 0.02% NaN₃) and incubate at room temperature for 20 min. Transfer oocytes quickly through three drops of 400 µL blocking solution to remove any residual detergent.
   NOTE: A 9-well clear glass spot plate works well for moving groups through multiple treatments on a single dish (steps 2.4-2.5).
8. Perform the remaining immunocytochemistry procedures in a humidified chamber, protected from light at room temperature. Use a medium-sized plastic container lined with damp paper towels and cover with aluminum foil.
9. Using the pipette apparatus, transfer oocytes to 30 µL blocking solution on the lid of a 96-well plate and place within the humidified chamber for 10 min.
   NOTE: Drops are placed in the circular indentions on the plate.
10. To label centromeres and spindle, using the pipette apparatus, transfer oocytes to 30 µL blocking solution containing anti-centromeric antigen (ACA, Crest) (1:30) and anti-acetylated tubulin (1:1000) and incubate for at least 1 h.
11. Rinse oocytes by transferring through three 30 µL drops of blocking solution for 10 min each.
12. Using the pipette apparatus, transfer oocytes to a 30 µL drop of blocking solution containing secondary antibodies complimentary to the antibodies used above (anti-human IgG 1:200, anti-mouse IgG 1:200) and incubate for 1 h.
13. Repeat the 3, 10-min rinsing steps in 30 µL blocking solution as described in step 2.11.
14. Transfer the oocytes, using the pipette apparatus, into 3-5 µL mounting medium containing DAPI (5.9 µg/µL) on a glass microscope slide.
15. Place 4 small drops (size of a pin head) of petroleum jelly on the corners of the cover slip to prevent crushing of the oocytes.
16. Gently place the cover slip petroleum-jelly side down onto the slide.
17. Seal coverslip edges with clear nail polish and allow to dry for 5 min.
18. Store slides at 4 °C, protected from light, until processing via confocal microscopy.
    NOTE: Ensure the refractive index of mounting medium, and coverslip, matches the microscope objectives being utilized. Recommended mounting materials have previously been described ¹⁵.
19. Image oocytes using a 40-63x objective on a confocal microscope, capturing small steps (≤ 1 µm) in the z-plane, optimized for the working objective, and image the entire region of the metaphase spindle.
    NOTE: Make sure to image all 3 channels based on specific secondary antibodies used in step 2.11. Image averaging ≥ 4 and a zoom of 3 are ideal for adequate resolution. A 1 µm step size provides clear visualization of microtubules and kinetochores in mouse oocytes. The procedure for image capture will vary from microscope to microscope. Please refer to the manufacturers confocal user guide for specific steps on how to configure microscope settings.
20. Identify kinetochore-microtubule attachment status using imaging software following steps 2.21-2.23 below.
    NOTE: The following steps describe this procedure using Image J (NIH) free downloadable software, however, alternate imaging software can be used. Attachment status types have previously been defined ¹⁶ .
21. Open image by dragging file into Image J tool bar.
22. In the opened z-series, make all channels visible (DNA, kinetochore, and spindle) by clicking "merge channels", available in the "Image" drop-down menu, under the "color" sub tab.
23. Using the toggle at the bottom of the image, move through each z-slice and determine kinetochore microtubule attachment. Detailed attachment descriptions have been previously described ¹⁶

3. Chromosome Alignment Challenge Assay

1. Using the pipette apparatus as described in step 2.3, transfer oocytes into a 100 µL drop of milrinone-free culture medium under oil and incubate oocytes for 7 h to allow sufficient maturation to metaphase of meiosis I (Met I) as previously described in step 2.3 ¹⁷.
   NOTE: Oocyte collection, microinjection, and maturation procedures were outlined previously ¹⁵. Refer to the attachment assay protocol in steps 2.1-2.2 for controls and for RNA microinjection concentrations.
2. Using same pipette apparatus, transfer oocytes to a center-well organ culture dish containing 700 µL culture medium containing monastrol [100 µM] in the center well and 400 µL H₂0 in the surrounding ring and incubate at 37 °C for 2 h.
3. Rinse out monastrol by transferring oocytes, using the pipette apparatus as above, through three drops of 100 µL CZB culture medium, and then transfer the oocytes to a new center-well organ culture dish containing 700 µL culture medium + MG132 [5 µM] for 3 h in the center ring. Add 400 µL H₂0 to the outside ring.
4. Fix the oocytes by transferring them, using the pipette apparatus, into a well of a clear glass spot plate containing 400 µL 2% paraformaldehyde in PBS for 20 min at room temperature.
5. After fixation, transfer the oocytes, using the pipette apparatus, into a new well of a clear glass spot plate containing 400 µL blocking solution and store at 4 °C until processed for immunostaining.
   NOTE: For storing oocytes at 4 °C, cover glass spot plate with parafilm to prevent evaporation.
6. Permeabilize and label oocytes via immunocytochemistry as described in steps 2.6-2.16.
7. Image oocytes using a 40-63x objective on a confocal microscope, capturing <1 µm steps in the z-plane making sure to image the entire region of the metaphase spindle.
8. To identify chromosome alignment status open image files using imaging software.
   NOTE: The following steps describe this procedure using Image J (NIH) free downloadable software, however, alternate imaging software can be used.
9. Drag image into image J control panel.
10. In the opened z-series, use the point tool (available by clicking the point tool icon on the Image J control bar) to mark points at the end of each spindle pole in their respective z-slice by placing the tool over the spindle pole in the image and clicking on the image. Add these points to the Region of interest (ROI) manager by pressing Command+t on the keyboard.
11. Determine the specific coordinates for each of the positions set in step 3.10 by highlighting the specific point in the ROI manager and clicking the measure button. This will provide a results table-containing x and y coordinates for each point. These will be the X1, Y1, and X1, Y2, points respectively.
12. Next, determine the true Z coordinate. This is done by multiplying the slice number of the specific z-slice in the z-stack in which the spindle pole was identified (i.e. slice #2 of 6) that each individual point is in by the stack thickness (i.e. 1 µm) (example: slice 2 x 1 µm = 2). These will be the Z1 and Z2 points respectively.
13. Determine spindle length using the Pythagorean theorem equation (a² + b² = c²) using the previously defined x, y, and z coordinates from steps 3.11-3.13. For each spindle the final length is equal to:
    √((X1-X2)²+(Y1-Y2)²+(Z1-Z2)²)
14. Determine the spindle midzone. This is calculated as one half the length of the spindle. Using the line tool in Image J, by clicking the line icon in the Image J tool bar, draw a line that is from one spindle pole to the spindle midzone. The length will be displayed on the Image J tool bar.
15. Determine chromosome alignment status by assessing chromosome distance from the spindle midzone using the line measurement tool. Using the line tool available by clicking the line icon in the Image J tool bar, draw a line from the spindle midzone to the chromosome. The length will be displayed on the Image J tool bar. Chromosomes greater than 4 µm from the spindle midzone are considered unaligned ¹⁸.

Results

After in vitro transcription high quality RNA will have an A260/A280 ratio of (1.8-2.2) and an A260/A230 ratio ≥1.7 when measured using a spectrophotometer. The picture in Figure 1 shows the migration of in vitro-produced RNA on a denaturing agarose gel after electrophoresis. A band that is smeared in pattern or a sample that has multiple sized bands can indicate contamination or degradation of the sample. Alternatively, multiple bands may i...

Discussion

Because of the rapid and increasing identification of human genetic variants associated with disease, it is essential that systems are established to evaluate their biological significance. Understanding protein function in human meiosis poses particular challenges because human oocytes are precious and rare and human sperm are not amenable to genetic manipulation. Mouse oocytes are a mammalian model system valuable for evaluating human meiotic gene function ^10,23

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL Seal-Rite PCR tube	USA Scientific	1602-4300
1 kb DNA Ladder	Thermo Scientific	SM0313
100 bp DNA Ladder	Thermo Scientific	SM0243
6X DNA loading Dye	Thermo Scientific	R0611
9-well glass spot plate	Thomas Scientific	7812G17
Agarose	Sigma Aldrich	A9539
Albumin from bovine serum	Sigma-Aldrich	A3294
Alexa-fluor-568 conjugated anti-mouse IgG	Thermo Scientific	A21050	1:200 dilution
Alexa-fluor-633 conjugated anti-human IgG	Thermo Scientific	A21091	1:200 dilution
Ampicillin	VWR	AA0356
Anti-vibration table	Technical Manufacturing Corp		any standard model
Anti-Acetylated Tubulin antibody	Sigma Aldrich	T7451	1:100 diution
Anti-centromeric (CREST) antibody	Antibodies Incorportated	15-234	1:30 dilution
Barrier (Filter) Pipette tips	Thermo Scientific	AM12635	Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors.
BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani)	Fisher Scientific	DF0445-07-6
BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani)	Fisher Scientific	DF0446-07-5
Capillary tubing	Sutter	B100-75-10
Center Well organ culture dish	VWR	25381-141
CO2 tank			For incubator
Confocal microscope	Zeiss		any standard model
Centrifuge (With cooling ability)	Thomas Scientific		any standard model
Cover Glass 11 x 22 mm	Thomas Scientific	6663F10
Coverslips	Thomas Scientific	6663-F10	thickness will vary for particular microscopes
DAPI	Sigma Aldrich	D9542
DEPC H20	Life Technologies	AM9922
Digital Dry Bath	Thermo Scientific	888700001
Easy A high fidelity cloning enzyme	Agilent	600400	For DNA cloning
Enzymes for linearizing pIVT	New England Biolabs		NdeI or KasI can be used
Ethidium Bromide	Thermo Scientific	155585011
Fluorescent Microscope			Any Fluorescent microscope may be used
Formaldehyde (37%)	Thermo Fisher Scientifc	9311
Formaldehyde RNA loading dye	Ambion	8552
Frosted Microscope Slides (Uncharged) 25X75 mm	Fisher Scientific	12-544-3
Full Length cDNA Clones			Can be obtained from any vendor that supplies open reading frame clones
Gel electrophoresis apparatus	Bio-Rad		any standard model
Glass Pasteur Pipets	Fisher Scientific	13-678-200
Globin Forward Primer for pIVT Construct			5'- GAA GCT CAG AAT AAA CGC -3'.  Can be purchased from any company that generates custom oligonucelotides
Globin Reverse Primer for pIVT Construct			5'- ATT CGG GTG TTC TTG AGG CTG G -3' Can be purchased from any company that generates custom oligonucelotides
Holding pipettes	Eppendorf	930001015	Vacutip
Humidified Chamber			Tupperware can be used
Illustra Ready-To-Go RT-PCR beads	GE Life Sciences	27925901
Incubator			any standard model with CO2 and water jacketed technology
Inverted Microscope	Nikon instruments		Any Standard model
Image J (NIH) Software	NIH		Image Analysis software
Lid of 96 well plate	Nalgene Nunc International	263339
Low Adhesion 0.5 mL microcentrifgue tube	USA Scientific	1405-2600
MacVector	MacVector		Sequence analysis software
MG132	Selleckchem	S2619
Microscope slides	Fisher Scientific	12-544-3
Millenium RNA Markers-Formaldehyde	Ambion	AM7151
Milrinone	Sigma-Aldrich	M4659	Resuspend in DMSO at 2.5mM
Mineral Oil	Sigma-Aldrich	M5310	Only used embryo-tested, sterile-filtered
Monastrol	Sigma-Aldrich	M8515	Resuspend in DMSO at 100 mM
Mouthpiece	Biodiseno	MP-001-Y
N2 tank			for antivibration table
Nail Polish; Clear			Any clear nailpolish can be used
NanoDrop Microvolume UV-Vis Spectrophotometer	Thermo Scientific		any standard model
NorthernMax 10X Denaturing Gel Buffer	Life Technologies	AM8676
NorthernMax 10X Running buffer	Life Technologies	AM8671
NuPAGE MOPS SDS Running buffer	Thermo Scnentific	NP0001
Organ Culture Dish 60x15mm	Life Technologies	08-772-12
Paraformaldehyde	Polysciences, Inc.	577773
PCR Thermal Cycler	Thermo Fisher Scientific	4484075
Petri Dish 139 mm	Thermo Fisher Scientifc	501V
Petri dish 35 mm	Thermo Fisher Scientifc	121V
Petri Dish 60 mm	Falcon BD	351007
Picoinjector	XenoWorks Digital Microinjector		any standard model
Pipette puller	Flaming-Brown Micropipette puller	Model P-1000
pIVT plasmid	AddGene	32374	Empty vector suitable for oocyte expression.
Pregnant Mare Serum Gonadotropin	Lee BioSolutions	493-10
QIAprep Spin Miniprep Kit	Qiagen	27104	purification of up to 20 uL of plasmid DNA
QIAquick PCR purification kit	Qiagen	28104
Quikchange II site directed mutagenesis kit	Agilent	200523	mutagenesis kit for insertions and deletions
Quikchange lightning multi-site directed mutagenesis kit	Agilent	210512	mutagenesis kit for single site changes
Scissors (Fine point)	Fine science tools	14393
Scissors (Medium point)	Fine science tools	WP114225
Seal-Rite 1.5 mL microcentrifuge tube	USA Scientific	1615-5500
Slide Warmer			any standard model
Spectrophotometer (Nanodrop)	Thermo Fisher Scientific	ND-ONE-W
Stereomicroscope			any standard model
Subcloning Efficiency DH5a Competent Cells	Thermo Fisher Scientifc	18265017
Syringe	BD Bioscienes	309623	1 ml, 27G(1/2)
T4 DNA Ligase	New England Biolabs	M0202L
T7 mMessage Machine high-yield capped RNA transcription kit	Life Technologies	AM1340
TritonX-100	Sigma-Aldrich	x-100
Tween-20	Sigma-Aldrich	274348
Tweezer (Fine point- Size 5)	Fine science tools	SN.743.12.1
UltraPure Dnase/Rnase-Free Distilled Water	Thermo Fisher Scientifc	10977015
UltraPure Ethidium Bromide 10mg/mL	Thermo Fisher Scientifc	15585011
UVP UV/White lite transilluminator	Fisher Scientific	UV95041501
Vectashield Mounting Medium	Vector Laboratories	H-1000