1. Use appropriate Data Collection Form to record necessary information

2. Take digital pictures with hand held camera 

1.  Obtain a Data Collection Form and use it to record the information you gather during the process
 

2. Take digital pictures with hand held camera
 

2.1 Types of Images Needed

• In The Field:  helpful shots include site situation,  field or landscape patterns of damage,  severity levels,  whole plant appearance. (A comparison to a healthy plant would be helpful).
• In the Office: visual image of  abnormalities present on foliage, stems, vascular systems, trunks, roots.
• Include visual images of any fungal structures or bacterial ooze droplets

The Sony Digital Camera has a number of internal settings which control certain qualities of the images you create.  The following settings should  work best for use with the Digital Diagnostic Program.
 

To change or check these settings:

• Turn the camera on by sliding down the "Power On/Off" switch
• Move the Play/Still/ Movie switch to the "Still" position
• In the lower right corner of the LCD screen (or the viewfinder), the word "MENU" should be highlighted in yellow.
• Press the center of the "Control Button" and a list of settings beginning with "Clock Set", "Beep"... should be displayed.
• Press the upward arrow on the edge of the "Control Button" to move up the list of options.  As you move up the list, each setting  will be highlighted in yellow and the current option for that setting will be displayed to the right of the highlighted selection.
• To change the setting, move the highlighted bar to the setting you  would like to change by using the up or down arrow on the edge of the "Control Button".  Then press the center of the "Control Button" to display the options for that particular setting.  Again use the up and down arrows on the edge of the "Control Button" to highlight the particular option you would like to set.  Press the center of the "Control Button" to execute that setting selection.

• Use the neck strap when the camera is being used.   When not in use, keep the camera in the case.
• Keep one battery charged at all times.
• The camera records on 3.5 inch floppy diskettes.  Be sure to have diskettes available.
• Turn the camera on by sliding down the "Power On/Off" switch.
• Move the Play/Still/ Movie switch to the "Still" position.
• Be sure to remove lens cover.
• To view the subject through the view finder as you would with a 35 mm camera, move the Finder/LCD switch to "FINDER".  Or to use the LCD screen to view the subject, move the switch to LCD.
• Frame the subject in the viewing screen (or viewfinder)
• Press the Shutter Button halfway down until the green light appears in the viewing area.
• Hold camera steady and press the Shutter Button down fully
• The image is captured immediately, but it does take a few seconds for the camera to automatically transfer the image to the diskette.

• For best resolution, move the Zoom Lever as wide as possible by holding lever toward the "W" and get as close to the subject as possible.
• Set the camera on "Auto Focus."  After the camera focuses with "Auto Focus", you can move the switch to "Manual Focus," and use the Focus Ring,  if a sharper picture is needed.
• If possible a light to medium gray background is desirable.
• The Spot Meter can be used to take a picture with the appropriate exposure for just the point you want to focus on.

• Start MGI Photo Suite software by clicking on the MGI icon on the desktop.
• Click on "File" from the menu bar, highlight "Get Photos From", then "Floppy Disk."
• When prompted, insert the 3.5 floppy disk which you used to record images using the hand held Sony camera and click "OK."
• Click on the individual thumbnails to enlarge and search for the images which best represent the problem.  Record the file names (or file numbers) and a description on the DDDI Sample Submission Log.
• The DDDI Sample Submission Log is a paper form used to hand record information about a particular sample submitted.  It will help you organize the images throughout the submission process.  Either of the following forms in Word 97 format can be used.  (Sample Submission Log Type 1)  or  (Sample Submission Log Type 2)
• You can leave the MGI Photo Suite running and just minimize the program window until your sample submission is complete, or you can click "File", then "Exit" from the menu bar.
• When exiting the program, a prompt will appear to ask you if you want to "Save Changes to Photo Album".  Choosing "Abandon" will keep your Photo Album clear for future use but not erase any of the images from your floppy diskette.

3.1 Selecting samples for microscopic images

• Look for symptoms on foliage, stems, trunk, roots.
• Many dieback symptoms are caused by  poorly functioning roots.
• Look for fungal bodies on foliage, stems, branches, trunk, roots.

• There are leaf spot samples and gall samples.
• There are problems other than leaf spots and foliage galls.
• Samples should be fresh, adequate in quantity, and representative, including early, mid, and late stages of  disease development.
• Information should be collected on: recent weather, chemicals/fertilizers used recently, soil conditions,  insects, animals, disease.

• Leaf Spots: enough to make 3-4 slide mounts.
• Microscopic diagnoses are most successful with foliage leaf spots and blights.

• Collect whole plants
• For large plants 3 or more feet tall, 1 plant is needed.
• For medium sized plants1-3 feet tall , 3-6 plants usually are needed
• For small plants 6-12  inches, 5-10 plants are needed.
• With seedlings less than 6 inches tall, 10-30 plants needed.
• With turf grass, a 7 by 7 inch square, 3 inch deep sample is needed. One half of the sample should be alive and healthy; one half should be damaged.

4.1 Using the Dissecting Microscope

• Place the specimen on the stage surface
• Make sure the Dimmer Switch is in the "Off" position
• Using the Illuminator Selector, select the light source (I=above, T=below, IT=both)
• Then Slowly turn the dimmer switch on to the desired level of illumination
• When viewing the specimen through the eyepieces, push the knob for the camera tube shutter "In"
• Adjust the two ocular lenses to your eyes to that only one image is seen
• Use Focusing Knob first to focus on the specimen at the lowest magnification
• Use the Zoom Control Knob to increase magnification from 10 to 60

• Images of any abnormal specks, dots, swellings or lesions when tissues are viewed at 10-40X

• Be sure the video camera is mounted on the microscope with the specimen to be photographed.
• On the computer, click on the FlashPoint Icon (FPG32).
• Be sure the knob for the Camera tube shutter is pulled all the way out.
• When image appears, position the part of the specimen to be photographed in the FlashPoint viewing screen, and do the final fine focus on the screen.
• Click the "Grab" option from the FlashPoint menu bar.
• Click on the "File" menu from the FlashPoint menu bar and choose "Save Image."
• A "Save in" box will appear at the top of the screen.  Select the drive or folder location to save the images (preferably the 3.5 floppy where the Sony camera images are already saved).  " Files of Type" should be set to JPG.
• Give the image a file name (no more than 8 characters of only letters or numbers)  plus the " .JPG" extension.  Click "Open" to save the image.
• To capture more images, click on  "Live" from the FlashPoint menu bar and repeat the above 5 steps.

5.1 Tape Mount Slides

• Use tape mount if fungal structures (spores) are present on the leaf surface.
• Press a piece of clear cellophane tape (about 1/2 to 3/4 inch long) onto the leaf surface.
• Remove the tape and place it (sticky side down) onto a drop of water (or cotton blue solution in water) on a slide.
• View the slide preparation.

• Place a drop of water on a glass slide.  Cotton blue stain may be used instead of water.
• Using dissecting microscope, focus on the fungal fruiting structures.
• Cut out a small section of the tissue containing the structures with a dissecting needle.
• Do not cut too large a section because you will not be able to view it clearly with the compound microscope.
• Place the cut out section in the water droplet.
• Cut several pieces and place all within the same water droplet to increase chances of clearly viewing the fungus.
• Place a cover slip over the water droplet.  Place one side of the cover slip against the water droplet and gently let the cover slip fall over the droplet.
• Tap with the blunt end of a dissecting needle to remove air bubbles.

6.1 Using the Compound Microscope

• Slowly turn on the illuminator switch
• Move the course focus knob so as to separate the stage from the objective lens as much as possible
• Place the prepared slide on the stage
• Rotate the objective so that the 4x lens is in place for viewing the specimen
• While observing from the side, use the coarse focus knob to lower the objective as far as possible
• Use the mechanical stage control knobs to move the slide on the stage so the slide is centered under the objective
• Adjust the ocular lenses to fit your eyes so that one image is seen
• Adjust light intensity as needed using the illuminator switch
• Make sure both field iris and aperture iris are fully open
• When viewing the specimen through the eyepieces, push the knob for the camera tube shutter "In"
• Use coarse focus knob, then the fine focus knob to get a sharp image
• To increase magnification rotate the 10x objective into place and focus using the fine focus knob
• To increase magnification rotate the 40x objective into place and focus using the fine focus knob
• Use Caution when adjusting the coarse focus knob as this can raise the slide into the objective
• When removing a slide from the stage, first raise the objective lens by turning the coarse focus knob
• When viewing a new slide, always start with the lowest power objective

• View  areas  that exhibited abnormal  structures under the stereo microscope.
• View other areas of  tissue damage.
• Make observations and images at 40, 100, and 400X  as appropriate.
• For most microscopic observations of  tissue,  make wet mount slide preparations.
• When leaves are especially thick or when fungal structures are suspected to be colorless, or when it  is  desirable to preserve the arrangements of spores present on foliage, a tape mount may be used.
• When fungal structures are suspected to be colorless or difficult to view, a stain for fungi is sometimes used. Acid Fuchsin or Cotton Blue are two commonly used fungal stains.

• Be sure the video camera is mounted on the microscope with the specimen to be photographed.
• On the computer click on the FlashPoint Icon (FPG32).
• Be sure the knob for the Camera tube shutter is pulled all the way out.
• When image appears, position the part of the specimen to be photographed in the FlashPoint viewing screen, and do the final fine focus on the screen.
• Click the "Grab" option from the FlashPoint menu bar to "snap the picture".
• Click on the "File" menu from the FlashPoint menu bar and choose "Save image."
• A "Save in" box will appear at the top of the screen.  Select the drive or folder location to save the images (preferably the 3.5 floppy where the Sony camera images are already saved).   "Files of type" should be set to JPG.
• Give the image a file name (no more than 8 characters of only letters or numbers)  plus the " .JPG" extension.  Click "Open" to save the image.
• To capture more images, click on  "Live" from the FlashPoint menu bar and repeat the above 5 steps.



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7. Complete and submit the DDDI form online

7.1 Sample Submission Process

• Connect to the World Wide Web and use browser to navigate to  http://www.dddi.org/aces.
• Enter username and password and click the Login button.
• Choose appropriate category under "Submit a Sample."
• Either select an existing client from the drop down box or complete the client information for a new client.
• Enter all pertinent information in the Sample Form. Remember the fields in red are required.
• At the "Sample Image 1," you will submit an image file.  To do this, click on the "Browse" button.  A "File Upload" or "Choose File" box will appear at the top of the screen.  In the "Look in" box, select the location of the image file (in many cases this will be the 3.5 floppy drive).  The "Files of type" box should  should be set to "All files"
• Click on the image file you want to attach as "Sample Image 1" and then click "Open." This will place the file name and location in the "Sample image 1" field.  Click "Preview" to display the image you just attached and make sure it is the image you intended to send.  Record this number on the Sample Submission Log.
• Enter a file Description for the image.  This is a required field describing the sample such as type of plant, magnification, etc. Record this description on the Sample Submission Log.
• Repeat the above 3 steps until the necessary number of images (up to 5) and descriptions  have been attached to the form.
• When all information and image fields are filled as necessary, Click on "Submit Form" at the bottom.
• When the "Information Submitted" screen appears, record the "Submission Number" on the Sample Submission Log.

7.3 Sample Submission Log (Type 2; Word 97 Document)

• Slowly dim the microscopes and turn them off.
• Make sure the power to the video camera is off by unplugging the power cord..
• Cover the microscopes with dust covers.