Observing and Counting Bacteria

PMB/MCB112

Bacteria are small.

Typical bacteria are 1-10 m long and 0.3-1 m wide.
Eukaryotic cells have a wide range of sizes:
10 m in diameter (yeast)
10 m wide and >100 m long (skeletal muscle)
up to 1 m long (neuronal axons)

Bright-field microscopy

Magnification is theoretically infinite, but RESOLUTION is limited by the

wavelength of light used to capture the image, and by the light-gathering ability
of the lenses. For a good microscope using visible light, the resolution limit
is ~0.2 m or 200 nm.

Total magnification = product of magnification by the objective

lens (10-100x) and the oculars (10x)
Resolving power (resolution) is the smallest distance between
two objects that allows them to be seen as distinct objects
Resolution is a function of the wavelength of light used to create
the image and the light-gathering ability of the objective,
or its numerical aperture (NA)

R=/2NA
If we use green light (540 nm) to illuminate a sample with a
high-resolution objective (NA=1.4), the resolution limit
is ~200 nm.
higher resolution means that the value of R becomes smaller!!

To see an object, we need contrast between the object

and the background.
In bright-field microscopy, objects absorb or scatter light more
than the surrounding material, so they look dark
against a light background.
BUT most bacteria dont absorb
or scatter enough light to be
clearly visible in bright-field.

Saccharomyces cerevisiae
Bakers yeast, 8-10 m

Create contrast by staining the sample

Gram stain a differential stain that can be used to distinguish

between different organisms in the same specimen.

Gram stain continued

Create contrast optically

Objects in a specimen have different (higher) refractive index
than the surrounding medium. Phase contrast and differential
interference contrast (DIC) microscopy use polarized light and
specialized lens systems to make this difference visible to our
eyes. No stains are needed. LIVE CELLS can be observed.
DIC

Phase contrast

Caulobacter crescentus, scale bar = 1 m

Transmission electron microscopy (TEM)

Uses electrons as radiation and electromagnets as lenses.
Wavelength of an electron is a function of the potential
difference through which it is accelerated.
1.23/V1/2
(where is in nm and V is in volts)
A typical TEM uses 50,000 V, so these electrons have
= .0055 nm (compared to green light = 540 nm)
However, electron microscope objectives have very small NA,
because they can only gather refracted electrons over
a very small angle .
The decrease in wavelength more than compensates for the
decrease in NA, so resolution in TEM is much better
than in light microscopy.
Resolution of TEM is 0.2 nm, 1000x better than light microscopes.

Whole-mounted bacteria viewed by TEM

Features NOT visible by light microscopy are resolved
by electron microscopy

Visualizing proteins within cells to understand cellular

organization
1) Immunofluorescence microscopy: an antibody that recognizes
a specific protein is covalently attached to a fluor.
Fluors are molecules that absorb light at one wavelength
(shorter/higher energy) and emit light at another
wavelength (longer/lower energy).
Fixed cells are treated with the antibody and observed under a
microscope. Light of a specific wavelength hits the sample
to excite the fluor, and light emitted by the fluor is detected.

fluorescein
isothiocyanate

2) Protein fusions to Green Fluorescent Protein (GFP)

Isolated from jellyfish Aequoria victoria
Three amino acid side chains in the protein react
covalently inside the barrel to form a fluor that
absorbs blue light and emits green light.
Unlike chemical fluors, GFP is a protein with a corresponding
gene.
Using recombinant DNA technology, we can connect the gene for
a protein of interest to the gene for GFP, express this
GFP fusion protein in living bacteria, and observe its
location using fluorescence microscopy.

Protein X

GFP

GFP imaging

Excite with blue light

GFP fused to a Caulobacter protein CtrA

GFP attached to Protein X

emits green light

We typically take two pictures of the bacteria, one using using

IF or GFP and the other using phase or DIC. We then
compare the photos sided-by-side or overlay them.
The photo that detects the presence and location of the protein
will not always show you the outline of the cell.

How do we know an IF or GFP experiment is showing us

the REAL location of the protein?
What can go wrong?
IF: antibodies sometimes recognize more than one protein
GFP: fusing GFP to a target protein can change the function
or location of the protein
What to do?
Gold standard: when both IF and a GFP fusion give you the SAME
result about a proteins location
Other experiments that increase your confidence are
IF: compare fluorescence signal in wild-type bacteria compared to
a strain lacking the target protein
GFP: make sure that the GFP fusion protein is functional by
making sure it complements a deletion of the target protein

Resolution of different types of microscopy

how do I calculate it?
During the class you will learn sizes of objectsyou should be able to figure out whether they can
be seen using light vs. electron microscopy
Stains vs. optical contrast-main point is that staining often kills
cells, so you cant always use stains when visualizing live
cells
Immunofluorescence vs. GFP
both will show you the locations of proteins within cells
In IF, you must fix the cells and you need an antibody
that recognizes your protein of interest
BUT you dont need to be able to genetically
manipulate the bacteria
In GFP, you dont need an antibody and can use living
bacteria, but you do need to be able to express
the fusion protein in your bacteria and ensure
that it is functioning like the wild-type protein

Counting bacteria
-are they growing? how fast?
-how many are we using in this experiment?
-how many are present in a diseased sample?

1) Direct microscopic count

requires a counting chamber

Depth of liquid in counting

chamber is 0.02 mm, so the
volume under the 25-square
grid is 0.02 mm3 = 0.02 l =
2 x 10-5 ml
Number of cells in the
25-square grid = number in
2 x 10-5 ml, so number per
1 ml can be calculated

2) Viable count or colony count

Counts number of cells in a sample capable of growing up
into a colony on solid medium.

ONLY living cells are counted

Disadvantages:
if you are counting cells from an environmental sample,
the medium may be suitable for some species but
not for
others
could take days/weeks for colonies to grow

3) Turbidity-indirect measurement
of bacterial numbers

Light

As bacteria in grow, they convert

nutrients into cell mass.
Prism
Incident light, I0
Filter
Sample containing
cells ( )
Unscattered light, I
Photocell (measures
unscattered light, I )
Spectrophotometer
Optical density (OD)
Log

I0
I

Cultures appear turbid or opaque

because light passing through
the sample is scattered by
bacteria.
The turbidity of a culture
(its optical density) is proportional
to the number of cells present.

Turbidity or optical density is proportional to cell number,

but the exact relationship is different for each species
due to cell size and shape.
You must generate a standard curve that relates OD to cell
number counted by an independent method, such as
a direct microscopic count.
Advantages:
rapid
no sample destroyed
Disadvantages:
does not distinguish between live and dead cells
does not distinguish between different kinds of cells