Microscopic analysis

Steve Leach Novozymes Biologicals

400X 7200X

Wastewater Biomass

Anatomy of a Microscope

Light is reflected by a mirror and focused through a condenser onto the stage where the sample is placed, through a hole in the stage. The objective lenses magnify the image and the eyepiece lenses magnify it further for the observer.

Stage

Objectives

Eyepiece

Coarse and fine Focusing knobs

Condenser

Light source

Brightfield View 100X

Phase contrast View 100X

Phase contrast Condenser

Helpful additions

Care and feeding of your scope

USE Dust Cover!! Only use lense paper on

objectives

Dilute glass cleaners if using

Use air to blow dust off

Turn light source up slowly

Off-set objectives when not in use

Centering telescope!!

Brightfield

Uses include:

Stained samples

Blood samples

Pathogens

Water sample

Light from a lamp is concentrated and directed onto the specimen. Most bacterial cells are difficult to see because of their lack of contrast from the surrounding medium.

FISH slide

Darkfield Usage

Uses include:

Density tests

Inorganic (filter)

Phase Contrast

Uses include:

Floc size

Higher Life forms

Filaments

Inerts

This type of observation is based on the principle that cells differ in refractive index from their surrounding medium which bends some of the light which passes through them.

Protozoa

Amoebas

Flagellates

Free-swimming ciliates

Crawling ciliates

Carnivore ciliates

Stalked ciliates

Suctorians

Amoeba

Slow movement

False feet

Feed on organics and bacteria

Compete with bacteria

Testate Amoeba

Flagellate

-Feed on bacteria -Very fast -5-10X bacteria size -Have two flagella

Free-Swimming Ciliates

-cilia covers entire shape -lower BOD present -Sufficient D.O. -W.E.V. shows health -Asexually & Sexually

CV courtesy of Wilhelm Foissner University of Salzburg Austria *Approx. 13,000 ciliates described

Crawling Ciliates

pH close to neutral Cirri cover portion Established floc

Foissner et al. 1991 Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems, Band I Bazerisches Landesamt für Wasserwirtschaft Munich, Germany

Carnivorous Free-Swimmer

Adequate D.O Low BOD Healthy biomass

Foissner et al. 1995 Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems, Band 4 Bazerisches Landesamt für Wasserwirtschaft Munich, Germany Lionotus feeding on ciliate

Suctoria

Low ammonia Presence of ciliates With or without stalk

Foissner et al. 1995 Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems, Band 4 Bazerisches Landesamt für Wasserwirtschaft Munich, Germany

Stalked Ciliates

Single vs. colonial Length of stalk Detached stalks

Stalk Ciliates

Foissner et al. 1992: Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems, Band 2 Bayerisches Landesamt für Wasserwirtschaft Munich, Germany

Rotifer

-Over 80% are female -Longer Sludge age -Low BOD -Sufficient D.O. -Tardigrade food* -1500 known species

Rotifer jaws

Nematode

Low D.O. Long Sludge age

Bristle worm

Tardigrade-”Water Bear”

Lorica With Eggs Adequate D.O. Low BOD Neutral pH

Typical industrial issues

Increase in organic loading

Increase in inorganic loading

Temperature (Inc. or Dec.)

pH swings

Low nutrients

Unknown dump

Production change

Process shutdown

Aeration & Mixing

Chemical trials

Issues cont.;

Seasonal shifts

Raw material changes

Solids handling and wasting

Industry specific

Poor sampling

Inexperienced operator

Equipment problems*

Cost cutting

Consultants

Problem #1

Refinery

ASU

Solids floating on surface of secondary

Eff. TSS violation

No lab testing available

1000X Phase

Solution

Problem

Denitrification

Increase wasting (reduce total inventory)

Monitor ORP in 2nd

Problem #2

Chemical

ASU- Pure oxygen

Scum on surface of secondary

Eff. TSS violation

No lab testing available

1000X Phase monocolonies

Solution

Problem

Extreme Low F/M

Institute Step-feed

Inf. COD monitoring

Purchase microscope

100X Phase

Problem #3

Paper Mill

ASB

Loss of COD reduction

No lab testing available

400X Phase

Solution

Problem

Enzyme addition for Pitch control

BioRemove 1100 addition to compensate for increased COD

400X Phase

Tools to solve problems

Microexams

Filament ID’s

KPR study

Flask Study

Data analysis

DOUR

SOUR

Nutrient testing

ORP

Compound analysis

MicroTrace

OdorTrak

NZB Tech. Service

Molecular analysis**

Daily Microexams (on site)

Should be performed at least 3X per week

Limited in scope

Find Funny Hungry Bugs

Floc Size, shape, density

Filaments Abundance, type

Higher life forms Presence, abundance, diversity

Bulk water Clean, containing inerts or bacteria

Objective progression

10X darkfield 10X Phase contrast 40X Phase contrast

40X Phase contrast 40X Phase contrast 100X Phase contrast w/oil immersion

When should a detailed microexam be performed?

When daily microexams show signs of change.

When treatment performance is poor or worsens.

If foaming problems occur.

When the plant is undergoing operational changes.

For plant confirmation and confidence

When everything is running smoothly*

Sampling

Collect in screw-cap, plastic sample bottles. Small amount of sample is needed (< 100 ml). Fill bottle only half full.

Observe shortly after collection for on-site analysis. Refrigerate and ship on ice for off-site analysis.

Sample mixed liquor from the effluent side of tank. If parallel streams, analyze sample from each stream.

If foam occurs, collect mixed liquor without foam contamination. If foam is to be analyzed, collect in screw cap bottle without mixed

liquor contamination.

Performing the exam

Place small drop on slide (3 if possible)

Place glass coverslip on top (not plastic)

Place slide & coverslip between paper and lightly pat to remove excess water* (nail polish can seal coverslip)

Observe under 10X magnification (provides wider view)

Increase magnification to 40X (change condenser setting)

If possible observe under 100X magnification (remember only this magnification requires oil immersion)

Make notes and draw pictures*

Clean any oil off objectives immediately

Place dust cover on microscope

40

India Ink Reverse Stain

India ink completely penetrates healthy floc particles.

During a nutrient deficiency, bacteria store cBOD in the form of exocellular polymeric material within the floc particle.

The stored material is not penetrated by India ink.

41

India Ink Reverse Stain (continued) Protocol Mix 1 drop of MLSS and one drop of India ink.

Observe under phase contrast microscopy.

Black or golden-brown areas have little or no stored-up cBOD.

White areas indicate cBOD and a nutrient deficiency.

Negative Positive

Succession of Protozoa and Relative Numbers of Bacteria

Organic Loading and Predominant Microlife

Condition Predominant GroupsHigh F/M ; low MCRT Flagellates, amoeba, small free-

swimming ciliates

Moderate F/M ; average MCRT Good diversity. Dominated by free-swimming and stalked ciliates

Low F/M ; high MCRT Stalked ciliates, rotifers, and higherinvertebrates.

Presenter

Presentation Notes

The general outline to be used along side of the scope

Microexam Sample

Olympus CX41 $3,600.00 (2011)

Olympus BX50 with Fluorescence attachment- $23,000.00 (1996)

Nikon Eclipse 80i- $47,500.00 (2011)

NOVOZYMES PRESENTATION 12/06/2012 49

References

Wastewater Microbiology A Handbook for Operators. Glymph, Tony. American Water Works, Denver, CO., 2005

Manual on the Causes and Control of Activated Sludge Bulking, Foaming and Other Solids Separation Problems. Jenkins, David and Richard, Michael G., and Daigger, Glen T. CRC Press, Boca Raton, 2004

Wastewater Microbiology: The Microlife. The Microlife Task Force of the Water Environment Federation, Schuyler, Ronald G. Chair. Water Environment Federation, Alexandria, VA., 2001

Fresh-Water Invertebrates of the United States, Protozoa to Mollusca 3rd ed. Pennak, Robert W., Wiley, New York, NY., 1989

Activated Sludge Microbiology Poster. Marshall, Richard and Richard, Michael. WWW.metcgroup.com