Plant Pathology LabPLPA 303

Sections 502-504Fall 2015

Rules and Expectations Attendance is MANDATORY

Must have university excused absence in order to make-up missed assignments and exams

Points will be deducted from worksheets for questions relevant to the days assignment, if un-excused

Cell phones need to be put away Must read, understand, and follow all safety rules as

described on your signed acknowledgement forms Must come prepared with lab coat and/or safety

goggles when needed

Lab Safety Wash your hands before and after lab Wipe down your work area before and after an

experiment No Food or Drink during lab Safety features of this lab or in near area:

Fire blanket (in hallway) Chemical shower (in hallway) Sharps containers (in back of class) Autoclave Trash (in back of class)

Grading and Assignments Worksheets (600 points)

12 worksheets will be assigned 50 points each

Fungal Identification Project (240 points) 6 unknowns will be provided (each 40 points) Identify in 3 weeks

Exam (240 points) Comprehensive

Participation and Safety Compliance (120 points) Arrive on time Pre-lab quizzes Bringing you goggles and lab coat Having close toed shoes

Grading and Assignments

Final grades A = 1080-1200 points B = 960-1079 points C = 840-959 points D = 720-839 points F = 000-719 points

Tips for Success in Lab Read your lab manual!

It will help you understand the material and perform your lab in a timely manner

Ask questions about things you don’t understand! You can ask before, during, or after class

Go to lecture! For the most part, material covered in lecture and lab

will overlap

Lab 1Introduction to Lab

Techniques and Basic Microscopy

What is Plant Pathology? Plant Pathology is the study of plant diseases What does this involve?

Agents that cause diseases Mechanisms by which diseases are caused Interactions between plants and their disease

causing agents Different methods that we can use to control or

prevent diseases The Disease Triangle

Susceptible Host Virulent Pathogen Conducive Environment

What is Disease? According to Webster…

“A condition of the living plant body or of one of its parts that impairs normal function and is typically manifested by distinguishing signs and symptoms

Southern leaf blight (Cochliobolus heterostrophuson) of maize 

What types of agents cause disease?Pathogens (Biotic)

Fungi, bacteria, viruses, nematodes, and parasitic plants

Non-living agents (Abiotic) Light, temperature, herbicides/pesticides,

salt stress, etc.

Signs vs. Symptoms Signs: The pathogen or its parts

seen on a host plant Ex. Fruiting body, bacterial cells,

etc.

Symptoms: The external and internal reactions of a plant as a result of a diseaseEx. Rotting, spotting, wilting, etc.

Introduction to Lab TechniquesSterile TechniquePathogen Isolation from Plant Material

Culture MediaMacroscopic ObservationsMicroscopic Observations

Sterile Technique Objectives of Sterile Technique

Prevent contamination of sterile culture media Prevent contamination of pure culture Prevent contamination of neighbor’s work

area What is a contaminant?

An undesired organism or substance

Sterile Technique cont. Tips for Avoiding Contamination

Do not open plate more then absolutely necessary Use sterile equipment Don’t breathe, cough, sneeze, or speak towards open

plates Methods of Sterilization

Chemical: 10% Bleach 70% Ethanol

Heat: Flame sterilizing Autoclave

Sterilization Methods Chemical Sterilization

To surface sterilize, spray chemical on area and wipe To sterilize equipment, dip tool in alcohol and pass through

flame (Cool before use!) Heat Sterilization

Flame Hold metal tool in upper tip of lower cone of the flame

until red-hot (Cool before use!) Autoclave

Uses superheated steam under pressurized conditions Typically: 121°C at 15 psi for 15-20 minutes

Sterile Hood Manipulates air flow to provide a sterile environment

Media Culture Mixture of nutrients which support microbial growth Complex Media

Contains undefined compounds Exp. PDA

Selective Media Specified to isolate only a target organism

Exp. PDA + Amp Differential Media

Differentiates between target organism and others which may grow on the media

Exp. MacConkey’s Agar

Macroscopic Observations General characteristics observed without the aid of a

microscope Bacterial (colony)

Texture (dry, mucoid, viscous) Color (opaque, translucent, iridescent) Shape (circular, irregular, filamentous) Pigment (yellow, red, white, etc.)

Fungal Mode of growth (filamentous vs. yeast-like) Presence of Spores? (Not all spores are macroscopic) Pigment (black, white, yellow, etc.)

Microscopic Observations Characteristics which require the use of a microscope Bacteria

Gram status (positive vs. negative) Motility Shape (Bacillus, Coccus, or Spiral) Grouping (Micro-, diplo-, staphlo-, strepto-)

Fungi Morphology of fruiting structure Branching pattern of mycelium Size and Shape of conidia (spores)

Basic Light Microscopy One of the most useful tools for trying to identify

the causal agents of plant diseases

Sometimes, observation of a specimen under a microscope will provide all the information needed to diagnosis the causative agent of a plant disease

Basic microscopy may also help determine that a certain pathogen is not the cause of a plant disease

Types of Light Microscopes

Dissecting Microscope

Compound Microscope

Moving your Microscope

When moving micrscopes: Always make sure one hand is on the

arm Always make sure the other hand is

under the base Make sure the cord is coiled behind the

microscope and is not at risk of becoming loose and falling

Guidelines for Use Keep microscopes clean Initial focus should be obtained with low power objectives

(10X) When using high power lenses, ONLY use the fine focus

adjustment Handle properly when carrying/transporting the microscope Make sure that your work area is well organized Proper use of oil immersion lens (100X only) When you are finished for the day:

Remove slides from the stage Failure to remove slide will result in automatic 5 point

deduction for this lab Put the low power objective in position Clean lens with lens paper

Adjustment of Eyepieces To prevent discomfort or fatigue, microscopes

need to be adjusted to users needs

Push or pull the eyepieces apart till you can see one distinct image with both eyes

Focus with right eye, then use sleeve on left to adjust the focus for the both eyes

Using Dissection Microscope Used to observe plant material, tissue,

nematodes, etc. The stage is flush to the base Light comes from below or above Magnification usually less then 40x Samples are moved by hand

The Compound Microscope

Calculating Magnification To calculate the power of magnification: multiply the

power of the ocular lens by the power of the objective

Using the Compound Microscope To increase the magnification:

Make sure that your sample is centered and in focus with lower power objective

Switch to the next higher power objective by turning the revolving nosepiece

If the light is not observable or the circle of light is not centered make sure the nosepiece is in the correct position

Use the fine focus to sharpen the image

Oil-Immersion Lenses The lower power lenses are called “high-dry” lenses and do

not require oil Oil-immersion lenses require a drop of oil on the slide to

produce a good image Why?

Lens opening are very small and the oil adjusts the refraction so that light is not wasted

Which lenses are Oil-immersion? Lens will usually have a black line around its side Usually only 100X for this class

Oil-Immersion Lenses cont.

Proper Use With the nosepiece turned halfway to the

100X lens, place a drop of oil on the slide, and then turn all the way

Use only the fine focus knob with this lens!

Immediately after use, clean the lens with lens paper

Microscopy cont.When finished using your microscope,

it is required that you: Clean your lenses and stageRemove your slides or other

specimen Turn off the lightsBundle the cord and tuck it behind

the stage

Today’s Lab Review safety protocols Select microscope from cabinet Follow appropriate handling protocols Using the prepared slide on desk, follow procedure

in Lab Manual to visualize microbe Complete Microscopy worksheet (to be handed in at

the beginning of the next class) Follow instructions to properly clean and store the

microscopes

Next Week: Bacteriology

Microscopy Worksheets Due! SymptomatologyBacterial Soft Rot Koch’s postulate