Plant Pathology Lab

PLPA 303
Sections 502-504
Fall 2015

Rules and Expectations

Attendance is MANDATORY
Must

have university excused absence in order to makeup 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
Sharps

shower (in hallway)

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
A =

grades
1080-1200 points

= 960-1079 points

= 840-959 points

= 720-839 points

= 000-719 points

Tips for Success in Lab

Read your lab manual!

Ask questions about things you dont understand!

It will help you understand the material and perform your

lab in a timely manner
You can ask before, during, or after class

Go to lecture!

For the most part, material covered in lecture and lab will
overlap

Lab 1
Introduction 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
Light,

agents (Abiotic)

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
disease
Ex.

Rotting, spotting, wilting, etc.

Introduction to Lab Techniques

Sterile

Technique

Pathogen
Culture

Isolation from Plant Material

Media

Macroscopic
Microscopic

Observations
Observations

Sterile Technique
Objectives

of Sterile Technique

Prevent

contamination of sterile culture media

Prevent

contamination of pure culture

Prevent

contamination of neighbors work area

What
An

is a contaminant?
undesired organism or substance

Sterile Technique cont.

Tips for Avoiding Contamination

Do not open plate more then absolutely necessary

Use sterile equipment

Dont 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:

121C 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.

Selective Media

Specified to isolate only a target organism

Exp.

PDA

PDA + Amp

Differential Media

Differentiates between target organism and others which may

grow on the media
Exp.

MacConkeys 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
Branching
Size

of fruiting structure

pattern of mycelium

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
Samples

usually less then 40x

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

paper

after use, clean the lens with lens

Microscopy cont.
When

finished using your microscope, it is

required that you:
Clean

your lenses and stage

Remove
Turn

your slides or other specimen

off the lights

Bundle

stage

the cord and tuck it behind the

Todays 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!

Symptomatology
Bacterial
Kochs

Soft Rot

postulate