Bacteria are Everywhere

By: Lauren Senter

Dr. Hamrick

STEP Program at Campbell University

Overview

Part 1– Places we find bacteria– Identification

Part 2– Control

Part 3– Detection in drug products

Places we find bacteria:

Hands Throat Nose Food

Hand Washing Experiment

This experiment shows how bacteria normally lives on your skin. When you wash your hands, you remove the surface bacteria that can make you sick but it will not kill or remove all the bacteria on your skin.

To do this experiment:– Make imprint of hand on an agar plate before

washing hands.– Then make an imprint of hand after washing hands

thoroughly with soap and water.– Incubate until the bacteria grows.

Bacteria in the Throat

For this experiment, we were able to look at and see the different types of bacteria in the throat.

To do this experiment:– Gently rub the back of

throat with a sterile cotton swab.

– Then rub the bacteria onto a rich media.

Bacteria in the Nose

In this experiment, we were looking for a specific kind of bacteria.

In this experiment:– Using a sterile cotton

swab, gently rub the inside of one nostril.

– Then rub the bacteria onto an agar plate and incubate for 2 to 3 days.

Mannitol Salt agar

Bacteria in Food

In this experiment, we were looking for different types of bacteria in the foods we might eat.

To do this experiment:– Grind up alfalfa sprouts into a fine liquid.– Plate dilutions of the liquid, and incubate to grow up

the bacteria. One of the plates was labeled TNTC (or too

numerous to count).The other plate had a total of 165 colonies– 82,500,000 bacteria in a gram of alfalfa sprouts.

Identifying Bacteria

When we find bacteria, there are a number of ways to find out what kinds of bacteria we have.

Gram positive Gram negative

Cocci (spheres)

Cocci (spheres)

rods

rods

E. coli Haemophilus influenzae

Cells in clusterscatalase positiveStaphylococci

Cells in chainscatalase negative

Streptococcus pneumoniae

Bacillus subtilis

Coagulase positiveStaphylococcus aureus

Coagulase negativeStaphylococcus epidermidis

Easy to grow

Hard to grow

Flow chart for identifyingmicroorganisms

Identifying Bacteria

Gram Staining is the first step in figuring out what kind of bacteria you are dealing with.

Gram Staining determines whether the bacteria is gram positive, which consists of a cell membrane and a thick cell wall, or gram negative, which consists of an inner membrane, a thinner cell wall, and an outer membrane.

Gram-positive Gram-negative

Cytoplasm Cytoplasm

Cellmembrane

Cellmembrane

PeptidoglycanPeptidoglycan

Outermembrane

Periplasmicspace

Don’t Mess Up!

It is important for the bacteria to be a pure culture before putting in a test tube or running any test.– Single colony purification

If you have a mixture of bacteria when you run biochemical test they might appear positive even if some bacteria are negative.

It will cause you to mess up when classifying the bacteria.

1

2

3

4

Catalase Test

Hydrogen peroxide is a good chemical for killing bacteria.

Catalase is a bacterial enzyme that converts hydrogen peroxide into water and oxygen.

To do this experiment:– Use a loop to remove a

little bit of bacteria from the plate and smear it onto a microscope slide.

– Add 1 drop of hydrogen peroxide. If the organism produces a catalase, rapid bubbling will occur.

– This test helps to recognize if the gram + bacteria is Streptococci or Staphylococci.

Coagulase Test We did a third test to determine if the bacteria were coagulase positive or coagulase negative.

Coagulase is a bacterial enzyme that clots (or coagulates) plasma products.

This test would tell us if the Gram +, catalase +, bacteria were Staphylococcus aureus or Staphylococcus epidermidis.

Gram positive Gram negative

Cocci (spheres)

Cocci (spheres)

rods

rods

E. coli Haemophilus influenzae

Cells in clusterscatalase positiveStaphylococci

Cells in chainscatalase negative

Streptococcus pneumoniae

Bacillus subtilis

Coagulase positiveStaphylococcus aureus

Coagulase negativeStaphylococcus epidermidis

Easy to grow

Hard to grow

Flow chart for identifyingmicroorganisms

Clinical sample

Control

Bacteria in the wrong place can make us sick, therefore we have several ways to eliminate or control bacteria.

Some of the ways we can control bacteria are:– Antibiotics– UV light

Antibiotics Antibiotics kill bacteria. Different bacteria show different susceptibilities to

different antibiotics. To do this experiment:

– Lay little discs with antibiotics in them on plates inoculated with bacteria.

– Incubate and you are able to see the different susceptibilities of the bacteria to the medicines.

Sensitive

Resistant

UV Light

UV light damages bacterial DNA. Different bacteria have different susceptibilities to UV Light.

To do this experiment:– Swab half of each of 4 plates

with a spore-forming bacteria, and the other half with a non-spore forming bacteria.

– Place one plate (marked 0 min) into the incubator. Place the remaining plates into the hood under the UV light with their lids off. Place the sunglasses over the plate labeled 5min.

– Leave the plates in the hood for the allotted time, then take them out and put them into the incubator.

Results

0min. :All bacteria grew. 1min. :Bacillus subtilis is

able to form spores. Spores are more resistant to UV damage, but some of the bacteria were killed. – Without the ability to

produce spores most of the bacteria in the lower part of the plate were killed by the UV light.

3min. :A little more of the Bacillus continues to die off. The non-spore forming bacteria is almost completely dead.

5min. :Most of all bacteria are dead except for where the sunglasses were sitting.The sunglasses protected both kinds of bacteria from the UV light.

Detection

To do this experiment:– Grow up Bacillus subtilis as your test bacteria. – Generate a growth curve and add small amounts

of bacteria to the media. – The plan was to then add small amounts of

Bacillus to a drug preparation and pass through the filter.

– Then add media to the filters and wait for the growth of the bacteria.

~117 ~12

~117 ~12

Steritest

Add drug preparation (containing bacteria) to the filter unit

Add media and incubate

Each filter unit in this test received 2.4 x 104 bacteria.

Sum Up

From doing these experiments, I have learned that bacteria are everywhere and sometimes they belong; sometimes they don’t!