Microbiology: WJEC A level Biology Unit 3 revision notes

Lesson 1 of 13

What are microorganisms and why do we study bacteria?

These notes cover topic 3.4, Microbiology, in WJEC A level Biology Unit 3: Energy, and the Environment. Each lesson follows the WJEC specification, and the exam tips are based on BioWelsh analysis of WJEC past papers.

A is a living thing that is too small to see clearly without a microscope. The main groups are bacteria, , fungi, protoctists and viruses. This topic focuses on bacteria: how we classify them, how we grow them safely in a laboratory and how we count them.

Bacteria matter far more than their size suggests. Together with fungi, they are the main , breaking down dead organisms and recycling the nutrients inside them. Some bacteria are , which they cause disease in humans, animals or crops. Many others are harmless or useful, such as the bacteria used to make yoghurt.

Bacteria reproduce asexually by . One cell copies its DNA and then splits into two identical cells. In ideal conditions some bacteria can do this about every 20 minutes, so a single cell can become millions of cells in less than a day. This rapid growth is why food spoils quickly and why careful technique matters in the laboratory.

Why bacterial numbers rise so quickly

1) 1 cell
2) 2 cells after one division
3) 4 cells after two divisions
4) 8 cells after three divisions
5) About 1 million cells after twenty divisions

Like all organisms, bacteria are named using the binomial system, with a genus name followed by a name, for example aureus. After the first time the full name is written, the genus can be shortened to its first letter, so Escherichia coli becomes E. coli. The name often gives a clue about the bacterium. cereus is , and cholerae causes .

Lesson 2 of 13

How are bacteria classified by their shape?

The simplest way to begin classifying bacteria is to look at their shape under a microscope. Almost every bacterium fits one of three basic shapes. Scientists then describe the shape more precisely by noting how the cells group together after they divide.

The three basic shapes of bacteria
Shape name	What it looks like	Example genus
(plural )	A sphere	,
(plural )	A rod	, Escherichia
	A curved or twisted cell, including rigid spirals and flexible corkscrews

Notice that these are three dimensional shapes. A is a sphere, not a circle, and a is a rod, not a rectangle. WJEC mark schemes reject 'round' or 'circular' for , because those words describe flat shapes.

can be described further by their arrangement. When a divides, the two new cells may stay stuck together. If they separate after every division they are found singly. If they stay in pairs they are . If they keep dividing in one direction they form chains, called . If they divide in several directions they form grape-like clusters, called .

The diagram below shows each shape and arrangement. Use it together with the notes underneath.

Eight labelled panels. Top row: a single coccus, diplococci in pairs, streptococci in a chain and staphylococci in a cluster. Bottom row: rod shaped bacilli, comma shaped vibrios with a flagellum, a rigid spiral spirillum and a thin flexible spirochaete. — The shapes and arrangements of bacteria. Original BioWelsh diagram.

The top row shows spherical bacteria, the . The only difference between the four panels is how the cells are grouped.
are always seen in pairs. form a line like a string of beads. form an irregular cluster like a bunch of grapes.
The bottom row starts with , which are cells.
A is a curved rod, shaped like a comma, often with a flagellum at one end. cholerae, which causes , has this shape.
A is a rigid . A is a thinner, flexible corkscrew. Both are shaped bacteria.

Drawings make shapes easy to learn, but real bacteria look less tidy. The photographs below were taken with a scanning electron microscope (SEM), which fires electrons at the surface of cells to produce a detailed three dimensional image. SEM images are black and white, so any colour is added afterwards by computer.

Greyscale scanning electron micrograph of many smooth rod shaped bacteria with rounded ends, with a 2 micrometre scale bar. — Scanning electron micrograph of *E. coli*, rod shaped bacteria. Photo: NIAID, Public domain.

Scanning electron micrograph, coloured purple by computer, showing a cluster of large spherical bacterial cells, with a 500 nanometre scale bar. — Colourised scanning electron micrograph of *Staphylococcus aureus* in a cluster. Photo: CDC / Matthew J. Arduino, Public domain.

Greyscale scanning electron micrograph of many short, curved, comma shaped rod bacteria packed together. — Scanning electron micrograph of *Vibrio cholerae*. Photo: Ronald Taylor, Tom Kirn and Louisa Howard, Dartmouth Electron Microscope Facility, Public domain.

Greyscale scanning electron micrograph of long, thin, tightly coiled corkscrew shaped bacteria tangled together. — Scanning electron micrograph of *Treponema pallidum*, a spirochaete. Photo: CDC / Dr David Cox, Public domain.

E. coli: smooth, cells with rounded ends. These are .
aureus: spherical cells packed together in a cluster. These are . The purple colour has been added by computer and has nothing to do with the .
cholerae: short rods that are curved like a comma. These are vibrios.
Treponema pallidum: long, thin, tightly coiled cells. These are .
The scale bars show how tiny bacteria are. Most are between 0.5 µm and 5 µm long, so thousands would fit across a millimetre.

Lesson 3 of 13

What is the structure of a bacterial cell wall?

Every bacterium is surrounded by a cell wall outside its cell membrane. The wall is made of , which is also called . is a three dimensional mesh made of polysaccharide chains cross- by short chains of amino acids.

It gives the cell its shape, which is why bacteria can be classified by shape.
It gives the cell strength.
It stops the cell from bursting when water enters by . Without the wall the cell would swell and burst, which is called .

Bacteria have two different types of cell wall. bacteria have a thick layer of with no outer membrane. bacteria have a much thinner layer of , covered by an outer membrane containing . is a large molecule made of lipid joined to polysaccharide.

The diagram below compares the two walls. Read it from the outside of the cell, at the top, to the cytoplasm, at the bottom.

Two labelled cross-sections. Gram-positive: a thick peptidoglycan layer above the cell membrane and cytoplasm. Gram-negative: an outer membrane with lipopolysaccharide chains, a thin peptidoglycan layer, then the cell membrane and cytoplasm. — Cell walls of Gram-positive and Gram-negative bacteria. Original BioWelsh diagram.

On the left, the wall has one very thick layer of directly outside the cell membrane.
On the right, the wall has three layers outside the cytoplasm: the cell membrane, then a thin layer of , then an outer membrane.
The wavy lines on the outer membrane represent . It is only found in bacteria.
Both types have a cell membrane made of a phospholipid bilayer. The difference is in the layers outside it.

Gram-positive and Gram-negative cell walls compared
Feature	Gram-positive	Gram-negative
layer	Thick	Thin
Outer membrane with	Absent	Present
Colour after	Purple	Pink or red
Effect of and	More easily damaged	Protected by the outer membrane
Examples	, ,	E. coli, Salmonella, cholerae

The structure of the wall explains how some work. Bacteria keep making and breaking links in their , especially when they grow and divide. stops the cross-links from forming, so the new wall is weak and the cell bursts as water enters by . , an in tears and saliva, bonds in . bacteria are more easily affected, because their is exposed. In bacteria the outer membrane protects the underneath.

Lesson 4 of 13

How does the Gram stain work?

Bacteria are almost colourless, so they are hard to see under a light microscope. The is a technique that colours bacteria so that they can be seen and sorted into two groups, and . The colour a bacterium ends up depends on the structure of its cell wall.

First a thin smear of bacteria is spread onto a clean glass slide using a sterile . It is left to air dry and then passed quickly through a Bunsen flame a few times. This is called . It kills the bacteria and sticks them to the slide so they are not washed off during staining.

The four stages of the Gram stain
Stage	Reagent	What it does	Gram-positive after this stage	Gram-negative after this stage
1		A purple dye that stains the of all bacteria	Purple	Purple
2		Acts as a , fixing the more firmly to the	Purple	Purple
3	Alcohol or acetone-alcohol	A . It washes out of cells with a thin layer	Purple	Colourless
4		A red that colours any cell that has lost the purple dye	Purple	Pink or red

The diagram below follows one cell and one cell through each stage.

Four boxes showing a Gram-positive and a Gram-negative rod after each reagent. Both are purple after crystal violet and iodine. After alcohol the Gram-positive rod stays purple and the Gram-negative rod is colourless. After safranin the Gram-negative rod is pink. — The four stages of the Gram stain. Original BioWelsh diagram.

At the start, both cells are colourless.
After and , both cells are purple. At this point you cannot tell them apart.
Stage 3 is the key step. The alcohol removes the purple dye from the cell, which becomes colourless, but the cell stays purple.
then stains the colourless cell pink. It also reaches the cell, but the dark purple hides it.

Why does the affect the two types differently? The thick layer of bacteria holds on to the and , so the dye is not washed out. bacteria have only a thin layer, and the alcohol dissolves the lipid in their outer membrane, so the dye is washed out easily.

The illustration below shows what a stained slide might look like when both types are present.

Circular microscope field containing purple spherical cells in clusters and separate pink rod shaped cells, with a 10 micrometre scale bar. — Illustration of a Gram-stained smear viewed with a light microscope. Original BioWelsh diagram.

The purple cells are spherical and grouped in clusters, so they are .
The pink cells are and found singly, so they are .
The scale bar shows 10 µm. Each is roughly 1 µm across, which is 1 × 10⁻³ mm.
A description of a bacterium on a slide should always include both its shape and its Gram reaction, for example ' '.

The photograph below is a real Gram-stained smear of a mixed culture, viewed at ×1000 with oil immersion. Real slides are messier than drawings, so it helps to know exactly what to look for.

Light micrograph of a Gram-stained smear. Dark purple spherical cells, many in clusters, are mixed with pink short rod shaped cells. Some areas are densely packed. — Gram-stained mixed culture of *E. coli* and *Staphylococcus aureus* at ×1000. Photo: Michael R Francisco, CC BY 2.0. Resized.

Find the dark purple cells first. They are tiny spheres, many of them in clusters. These are , aureus.
Now find the pink or red cells. They are short rods. These are , E. coli.
Some areas are crowded where the smear was spread too thickly. Look at the thinner areas, where individual cells can be seen clearly.
Write your answer in the form ' in clusters and ', giving both the colour result and the shape.

Lesson 5 of 13

How are microorganisms cultured in the laboratory?

To study bacteria we need a large number of them, so we grow them in a . A is a mixture that provides everything the bacteria need to grow. Growing bacteria in this way is called culturing.

is a liquid . It is useful for growing large numbers of bacteria and for making .
is a made solid with , a jelly extracted from seaweed. It is poured into Petri dishes to make , or set at an angle in a tube to make an slope. On , each bacterium that lands on the surface grows into a visible .

Ways of describing a culture medium
Type of medium	What it means	Example
Defined	Every ingredient and its exact amount is known	A made only from glucose, named salts and water
	Contains ingredients whose exact chemical make-up is not known	A containing yeast extract or beef extract
	Allows only certain bacteria to grow	A containing an , so only bacteria grow
Complete	Contains all the chemicals needed to support growth	for most common bacteria

The illustration below shows a plate after .

Illustration of a Petri dish of yellow nutrient agar with about sixty separate round white colonies scattered across the surface. — Bacterial colonies growing on a nutrient agar plate. Original BioWelsh diagram.

Each white dot is a , not a single bacterium.
A is a group of genetically identical cells that grew by from one bacterium, or one fungal spore.
Because each started from one living cell, counting tells you how many living cells were spread onto the plate. This idea is used in the later in the topic.

The photograph below shows a real . Bacteria were spread across the plate in lines, a method called streaking. Where many bacteria were spread, the have joined into lines. Towards the end of the streaks, where fewer bacteria were left on the , separate round can be seen, each grown from a single cell.

Photograph of a Petri dish on a dark background. Yellow bacterial growth forms continuous streaked lines, with separate small round colonies near the ends of the streaks. — A streaked agar plate with bacterial colonies. Photo: NOAA Ocean Explorer, Public domain.

Lesson 6 of 13

What conditions do bacteria need to grow?

Bacteria grow well only when their conditions are right. Each has a of conditions in which it can grow and an optimum within that . The main factors are nutrients, temperature, pH and oxygen.

Conditions needed for bacterial growth
Factor	Why it matters	Typical requirement
Carbon and energy source	Carbon is needed to make organic molecules, and of the source releases energy	Usually glucose
Nitrogen source	Needed to make amino acids, proteins and nucleic acids	Organic molecules, or inorganic ions such as
	Vitamins and mineral ions are needed in small amounts for metabolism	Vitamins such as biotin, and ions such as Na⁺, Mg²⁺, Cl⁻, SO₄²⁻ and PO₄³⁻
Water	Needed for chemical reactions and to dissolve nutrients	Supplied by the
Temperature	Bacterial metabolism is controlled by	Most bacteria grow between 25 °C and 45 °C; human have an optimum near 37 °C
pH	pH affects activity	Most bacteria prefer slightly alkaline conditions, about pH 7.4; fungi prefer neutral to slightly acidic conditions
Oxygen	Some bacteria need it for , but it is toxic to others	Depends on the (see below)

Temperature and pH matter for the same reason they matter in the topic. All of a bacterium's metabolic reactions are catalysed by . Too cold and the work slowly. Too hot, or at the wrong pH, and the are denatured, so the bacteria stop growing or die.

Bacteria grouped by their need for oxygen
Group	Relationship with oxygen	Example
	Must have oxygen to grow, because it relies on	Mycobacterium tuberculosis
	Grows best with oxygen, but can survive and grow without it using	E. coli
	Cannot grow when oxygen is present, because oxygen is toxic to it	Clostridium

A simple way to see these groups is to grow bacteria in tubes of nutrient . Oxygen from the air dissolves at the surface, so there is plenty of oxygen at the top and almost none at the bottom.

Three test tubes of nutrient medium. Tube A has growth only at the surface, labelled obligate aerobe. Tube B has growth throughout, densest near the top, labelled facultative anaerobe. Tube C has growth only at the bottom, labelled obligate anaerobe. — Where bacteria with different oxygen requirements grow in a tube of medium. Original BioWelsh diagram.

In tube A, the bacteria grow only at the surface, where there is the most oxygen. These are .
In tube B, the bacteria grow all the way through the tube but most densely near the top. They can grow without oxygen but grow best with it. These are .
In tube C, the bacteria grow only at the bottom, where there is no oxygen. These are .

Lesson 7 of 13

What is aseptic technique and why is it needed?

, also called sterile technique, is a set of laboratory practices that keeps apparatus and cultures free of unwanted . It has two aims, and it helps to learn them as a pair.

To stop the culture being contaminated by from the environment, such as bacteria from the air, the bench or your skin.
To stop the environment, including the people doing the work, being contaminated by the being cultured. Even harmless bacteria might mutate, and a culture could be contaminated with a .

The diagram below shows the whole process in order. The notes under it explain the reason for each step, because exam questions usually ask why a step is done, not just what it is.

Eight numbered boxes in order: sterilise, prepare the bench, flame the loop, inoculate, tape the lid, incubate, observe and count, dispose. — Aseptic technique from start to finish. Original BioWelsh diagram.

Sterilise the equipment and before use, usually in an at 121 °C for 15 minutes. An works like a large pressure cooker, and the high-pressure steam kills all , including spores. Plastic equipment that would melt is sterilised commercially using gamma radiation.
Wipe the bench with disinfectant before and after the work, and work close to a lit Bunsen burner. The rising hot air carries airborne upwards, away from the open culture.
Heat the wire in the flame until it glows red hot, then let it cool before use. This kills on the , and cooling stops the hot from killing the bacteria being transferred.
When transferring bacteria, remove the bottle cap with your little finger and keep holding it, pass the neck of the bottle through the flame, and lift the lid of the Petri dish only slightly. Flaming the neck creates an upward air current and kills at the opening. Lifting the lid only a little reduces the chance of airborne landing on the .
Secure the lid with two short strips of tape, but do not seal it all the way round. Full sealing would stop oxygen getting in and could create anaerobic conditions, which would favour the growth of anaerobic bacteria, including dangerous .
the plate upside down at about 25 °C. Upside down stops condensation dripping onto the . 25 °C is used instead of 37 °C because 37 °C is the ideal temperature for many human .
Once , do not open the Petri dish. Observe and count the through the lid.
the used plates, or seal them in autoclavable bags, before disposal. This kills the cultures so they cannot contaminate anyone.

The photograph below shows one of these steps in a real laboratory. A wire is being heated in a Bunsen burner flame until it glows red hot, while the culture tube is held ready in the other hand. The must then be left to cool for a few seconds, because a hot would kill the bacteria it touches.

Photograph of a person in a lab coat holding a wire inoculating loop in a Bunsen burner flame while holding a culture tube in the other hand. — Flaming a wire inoculating loop in a Bunsen burner flame. Photo: Deamit, CC BY 4.0. Resized.

Lesson 8 of 13

How can we measure the growth of a bacterial population?

Measuring how many bacteria are present is important in many jobs. Environmental health officers test food, water companies test drinking water and food manufacturers check their products are safe. There are two kinds of count, and the difference between them is examined often.

Viable counts and total counts
Type of count	What it counts	Method
	Living cells only	, plating and counting
	Living and dead cells together	Counting cells with a special counting slide under a microscope, or measuring

A relies on the fact that only a living cell can divide to form a . Dead cells do not form , so they are not counted.

. As bacteria multiply in a , it becomes cloudier. A measures how much light passes through the sample, and the reading is compared with a calibration graph to estimate the number of cells. This is quick, but it gives a , because dead cells make the cloudy too.

Lesson 9 of 13

How do serial dilution and colony counting work?

A sample of milk or pond water can contain millions of bacteria in every cubic centimetre. If you spread it straight onto an , the would grow into one another and could not be counted. The answer is to dilute the sample in a series of steps, which is called .

In a tenfold , 1 cm³ of the sample is added to 9 cm³ of sterile water and mixed. This new tube is ten times more dilute, a 10⁻¹ . Then 1 cm³ of that tube is added to another 9 cm³ of sterile water, giving a 10⁻² , and so on. A hundredfold can be made in the same way by adding 0.1 cm³ to 9.9 cm³.

The diagram below shows followed by plating. Follow it from left to right.

Six test tubes from the original culture to a 10 to the minus 5 dilution, with 1 cubic centimetre transferred into 9 cubic centimetres of sterile water each time. Below, agar plates from each dilution: too many to count, clumped colonies, 216 colonies, 23 colonies and 2 colonies. — Serial dilution, plating and counting colonies. Original BioWelsh diagram.

Each red arrow shows 1 cm³ being moved into the next tube, which already contains 9 cm³ of sterile water.
The colour of the liquid gets paler from left to right, showing the bacteria becoming less concentrated. Each tube is ten times more dilute than the one before.
0.1 cm³ from each tube is spread onto its own sterile and the plates are .
The 10⁻¹ and 10⁻² plates have so many that they merge together, which is called . They cannot be counted accurately.
The 10⁻³ plate has 216 separate , which can be counted accurately.
The 10⁻⁴ and 10⁻⁵ plates have too few to give a reliable estimate, because a small random difference would have a big effect on the result.

Worked example: number of bacteria in the original sample

Number of bacteria per cm³ = number of ÷ ( × volume plated) 216 ÷ (10⁻³ × 0.1 cm³) Choose the plate with separate, countable : the 10⁻³ plate with 216 . Account for the : the plate came from a 10⁻³ , so the original sample was 1000 times more concentrated. 216 ÷ 10⁻³ = 216 × 1000 = 216 000. Account for the volume plated: only 0.1 cm³ was spread, so divide by 0.1, which multiplies by 10. 216 000 × 10 = 2 160 000 Answer: 2.16 × 10⁶ bacteria per cm³ (2.2 × 10⁶ to 2 figures) Two mistakes are common: forgetting that only 0.1 cm³ was plated, and multiplying by the instead of dividing by it. A quick check is that the answer must be much bigger than the number of you counted.

A relies on an important assumption: that each grew from one single living bacterium. If two cells land very close together they may grow into one , so the count can be an underestimate. This is also why plates with are not used.

Lesson 10 of 13

What does a bacterial growth curve show?

If bacteria are added to a fresh, closed culture, such as a flask of , and counted at regular intervals, the numbers change in a predictable pattern called a growth curve. Because the numbers become enormous, the y axis usually shows the of the number of bacteria.

A to base 10 tells you the power of ten. For example, of 1 000 000 is 6, because 1 000 000 = 10⁶. Using numbers lets values from 100 to 100 000 000 fit on one sensible scale, and it turns a doubling into a straight line.

The graph below shows the four phases of growth.

Graph of log10 number of bacteria per cubic centimetre against time in hours. The curve is flat for about 3 hours (lag phase), rises steeply to about 9 hours (log phase), levels off until about 16 hours (stationary phase) and then falls (death phase). — The phases of a bacterial growth curve. Original BioWelsh graph.

, from 0 to about 3 hours: the numbers hardly change. The bacteria are adjusting to the new , taking up water and making the they need, so they are not yet dividing much.
Log or , from about 3 to 9 hours: the line rises steeply and almost straight. The bacteria are dividing at their fastest possible rate because nutrients are plentiful and there is little waste.
, from about 9 to 16 hours: the line levels off. Nutrients are running out and toxic waste is building up, so the rate at which new cells are made equals the rate at which cells die.
, after about 16 hours: the line falls. Cells now die faster than new cells are made, because nutrients are used up and waste has built up to toxic levels.

Lesson 11 of 13

How do we calculate generation time and the number of generations?

In the , the bacteria divide by at a steady, maximum rate. The is the time between one division and the next, so it is also the time the takes to double. For this reason it is also called the . The shorter the , the faster the grows. If a graph shows the actual numbers of bacteria, you can read the straight off it: find the time at which one count has doubled.

Each generation doubles the number of cells. After one generation there are 2 times as many cells, after two generations 4 times as many, after three generations 8 times as many, and after n generations 2ⁿ times as many. This gives the equation N = N₀ × 2ⁿ, where N₀ is the starting number of cells and N is the number after n generations.

Exam questions usually work the other way round. They give a graph of of the number of bacteria and ask for the number of generations or the . Taking logs of N = N₀ × 2ⁿ gives log N = log N₀ + n × log 2. Because 2 = 0.301, the number of generations is the change in log number divided by 0.301. WJEC gives the equation in the question. It is sometimes written as 3.3 × (log N − log N₀), which is the same thing, because 1 ÷ 0.301 is about 3.3.

The equations used for population growth calculations
What you want	Equation
Number of generations, n	n = (log N − log N₀) ÷ 0.301, or n = 3.3 × (log N − log N₀)
()	= time taken ÷ n
Rate of growth	Generations per hour = n ÷ time taken in hours

Choose two points in the , where the line on a log graph is straight.
Read the time and the number of bacteria at each point.
Subtract to find the change in log number and the time taken.
Divide the change in log number by 0.301 to find the number of generations.
Divide the time taken by the number of generations. Convert hours to minutes first if the answer is wanted in minutes.

Approximate generation times of some bacteria in good conditions
Bacterium	Approximate generation time
Escherichia coli	20 minutes
aureus	30 minutes
Mycobacterium tuberculosis, which causes tuberculosis	15 to 20 hours

Lesson 12 of 13

Specified practical: counting bacteria in fresh and stale milk

The specification requires you to investigate the numbers of bacteria in fresh and stale milk using , plating and counting . It brings together everything in this topic: , , , calculations and log scales.

Fresh milk contains relatively few bacteria. As milk ages, bacteria use the lactose in it, divide and increase in number, which is why old milk tastes sour. Counting the bacteria in milk of different ages shows this growth.

Specified practical

Investigating the number of bacteria in fresh and stale milk

Unit 3, section 4, specified practical 1 (BioWelsh entry number)

Aim To compare the number of bacteria in fresh and stale milk by making , plating them on and counting the that grow.

Five panels in order: milk bottles of different ages; a rack of test tubes for serial dilution; a spreader over an agar plate; an incubator at 25 °C holding plates; an agar plate with colonies marked as they are counted. — The milk practical at a glance. Original BioWelsh diagram.

Equipment

Milk samples of different ages
Milk samples of different agesAbout 5 cm³ of eachFresh milk and milk kept 2, 4, 6 and 8 days, all from one batch.
Sterile water in test tubes
Sterile water in test tubes9 cm³ per tubeDilutes each sample tenfold at each step.
Sterile pipettes
Sterile pipettes1 cm³ and 0.1 cm³Transfer exact volumes without adding .
Sterile nutrient agar plates
Sterile platesThree per Each living bacterium grows into a .
Sterile spreader
Sterile spreaderSpreads the sample evenly so are separate.
Bunsen burner and disinfectant
Bunsen burner and disinfectantThe updraft carries away; disinfect the bench before and after.
Adhesive tape and marker pen
Adhesive tape and marker penTwo strips of tape on each lid; label plates on the base.
Incubator
25 °CGrows bacteria without favouring human .
Colony counter or marker pen
counter or marker penMark each as it is counted so none is counted twice.
Autoclave
Kills all on used plates before disposal.

Safety

Bacteria cultured from milk, possibly including RiskInfection if cultures are released or plates are opened.Control, at 25 °C, tape lids without sealing, never open plates after , before disposal.
Bunsen burnerRiskBurns or fire, especially near alcohol used for spreaders.ControlKeep alcohol away from the flame, tie back long hair, use the safety flame when not heating.
Spilt culturesRiskContamination of the bench, skin or clothing.ControlCover with disinfectant and paper towels, tell the teacher, wash hands afterwards.

Variables

Independent variableAge of the milk

Milk from the same batch tested when fresh and after 2, 4, 6 and 8 days at room temperature.

Using the same batch any difference is caused by the age of the milk and not by milk from different sources.

Example: Fresh milk contained about 3.8 × 10⁴ bacteria per cm³, while 8 day old milk contained about 9.1 × 10⁸.

Dependent variableNumber of bacteria per cm³ of milk

Calculated from the number of on three plates, divided by the and by the volume plated.

This is a , so it measures only living bacteria.

Controlled variablesIncubation temperature, Incubation time, Type and volume of agar, Volume plated

temperature

All plates at 25 °C.

Temperature affects how fast bacteria grow, so a different temperature would change the number and size of .

time

All plates for 48 hours.

A longer time could let more become visible or merge together.

Type and volume of

The same , poured to the same depth in every plate.

Different nutrients could allow different numbers of bacteria to grow.

Volume plated

0.1 cm³ of each spread on every plate.

A larger volume would contain more bacteria and give more .

Control experimentBoiled and cooled milk

Plated in exactly the same way as the other samples.

This separate test should give no , showing that on the other plates come from bacteria in the milk and that the prevented contamination.

Control experiment or controlled variable?

A is kept the same for every plate, such as the temperature. A is a separate set of plates, here boiled and cooled milk, that shows whether come from the milk or from contamination. Do not use one term for the other.

Method, step by step

1)
Wipe the bench with disinfectant, wash your hands and work next to a lit Bunsen burner.
Why?
This reduces contamination of the samples from the bench and the air.
2)
Mix the first milk sample well. Using a sterile pipette, add 1 cm³ of milk to 9 cm³ of sterile water and mix. This is the 10⁻¹ .
Why?
Mixing makes sure bacteria are spread evenly, so 1 cm³ is representative of the whole sample.
3)
Using a fresh sterile pipette each time, transfer 1 cm³ from each tube into the next tube of 9 cm³ sterile water to make dilutions down to 10⁻⁶ or 10⁻⁷.
Why?
Each transfer dilutes the sample ten times. A fresh pipette stops bacteria being carried over from a more concentrated tube.
Serial dilution, plating and counting colonies. Original BioWelsh diagram.
Open full size
4)
Label the base of three for each with the milk age, the and the date.
Why?
The base is labelled because lids can be swapped. Three plates allow a to be calculated.
5)
Lift the lid of a plate only slightly and add 0.1 cm³ of the . Spread it evenly with a sterile spreader.
Why?
Lifting the lid slightly reduces airborne contamination. Even spreading gives separate .
6)
Secure each lid with two strips of tape without sealing it all the way round, and the plates upside down at 25 °C for 48 hours.
Why?
Full sealing could create anaerobic conditions that favour . Upside down stops condensation dripping onto the .
Aseptic technique from start to finish. Original BioWelsh diagram.
Open full size
7)
Repeat steps 2 to 6 for the milk of each age.
Why?
This gives results for fresh milk and progressively staler milk.
8)
As a , set up plates using milk that has been boiled and cooled.
Why?
Boiling kills the bacteria, so no should grow. This shows that come from bacteria in the milk and not from contamination.
9)
After , do not open the plates. For each milk age, choose the with separate that can be counted, count the on all three plates and calculate the .
Why?
Crowded plates give an underestimate because merge. Plates with very few give an unreliable estimate.
Bacterial colonies growing on a nutrient agar plate. Original BioWelsh diagram.
Open full size
10)
Calculate the number of bacteria per cm³ of milk, then its , and plot a graph against the age of the milk.
Why?
The numbers cover a huge , so values fit on one scale.
11)
all plates and used equipment before disposal.
Why?
This kills the cultures so that no one is exposed to them.

Results and analysis

Example data

counted and bacteria per cm³ in milk of different ages (0.1 cm³ of each plated, 48 hours at 25 °C)

Age of milk / days	counted	on plate 1	on plate 2	on plate 3		Bacteria per cm³ of milk	bacteria per cm³
0 (fresh)	10⁻²	34	41	38	37.7	3.8 × 10⁴	4.6
2	10⁻³	52	47	60	53.0	5.3 × 10⁵	5.7
4	10⁻⁵	29	35	26	30.0	3.0 × 10⁷	7.5
6	10⁻⁶	64	71	58	64.3	6.4 × 10⁸	8.8
8 (stale)	10⁻⁶	88	95	91	91.3	9.1 × 10⁸	9.0

Graph on graph paper of log10 number of bacteria per cubic centimetre of milk against age of milk in days. Points: 4.6 at day 0, 5.7 at day 2, 7.5 at day 4, 8.8 at day 6 and 9.0 at day 8, joined by straight lines. — Number of bacteria in milk of different ages, on a log₁₀ scale. Original BioWelsh graph.

What the results show

The number of bacteria per cm³ rose from 3.8 × 10⁴ in fresh milk to 9.1 × 10⁸ in 8 day old milk. Each rise of 1 on the ten times more bacteria, so this is an increase of more than 20 000 times.

Between day 0 and day 6, values rose steeply from 4.6 to 8.8. The bacteria were dividing rapidly by while lactose was plentiful, like the of a growth curve.

Between day 6 and day 8 the values only rose from 8.8 to 9.0. The was entering the as nutrients ran short and acid built up.

Repeat plates were close, for example 64, 71 and 58 at day 6, so the are fairly reliable. The boiled milk produced no .

Worked calculations

at day 4 = (29 + 35 + 26) ÷ 3 = 30.0

Bacteria per cm³ at day 4 = 30.0 ÷ (10⁻⁵ × 0.1) = 30.0 × 10⁵ × 10 = 3.0 × 10⁷

(3.0 × 10⁷) = 7.48, which is 7.5 to 1 decimal place

Evaluation

Each colony is assumed to come from a single bacterium, but two bacteria close together can grow into one colony.

Effect on resultsThe count is likely to be an underestimate of the true number of living bacteria.

ImprovementMix each thoroughly and spread samples evenly, and count only plates with well separated .

Only living bacteria that can grow on nutrient agar at 25 °C form colonies.

Effect on resultsBacteria with different requirements, including some that need no oxygen, are not counted.

ImprovementUse a of or conditions if a more complete count is needed.

Errors in measuring small volumes are multiplied by every step of the serial dilution.

Effect on resultsA small error in an early transfer makes all later dilutions inaccurate.

ImprovementUse accurate graduated pipettes or micropipettes and a fresh sterile tip for each transfer.

Only three plates are counted for each milk age.

Effect on resultsAn unusual plate has a big effect on the .

ImprovementUse more replicate plates and check for anomalous counts before calculating the .

Going further

Why log₁₀ values are used

The numbers from about 10⁴ to 10⁹, which is far too wide to plot clearly on an ordinary scale.
of a number is its power of ten, so 10⁴ becomes 4 and 10⁹ becomes 9.
Each increase of 1 on a ten times more bacteria.
Your calculator's log button gives directly. Give values to 1 decimal place unless told otherwise.

Fresh milk compared with stale milk

Fresh milk has relatively few bacteria, so a low such as 10⁻² gives countable plates.
Stale milk has many more bacteria, so much higher dilutions, such as 10⁻⁶, are needed before are separate.
In fermented milk drinks, bacteria such as Lactobacillus convert lactose into , which lowers the pH and sours the milk.

Model answer: describe the method

Describe how you would carry out a to estimate the number of bacteria in a sample of stale milk.

Original practice response · 6 indicative marks

Working aseptically next to a Bunsen burner on a disinfected bench, mix the milk and use a sterile pipette to add 1 cm³ of milk to 9 cm³ of sterile water to make a 10⁻¹ .
Using a fresh sterile pipette for each step, repeat the transfer of 1 cm³ into 9 cm³ of sterile water to make a of dilutions, for example down to 10⁻⁷.
Spread 0.1 cm³ of each onto sterile plates with a sterile spreader, lifting the lid only slightly, using three plates for each .
Tape the lids with two strips of tape and the plates upside down at 25 °C for 48 hours.
Choose the whose plates have separate that can be counted, count the on each plate and calculate the .
Calculate the number of bacteria per cm³ by dividing the number of by the and by the volume plated, for example 30 ÷ (10⁻⁵ × 0.1) = 3.0 × 10⁷ per cm³.

What the answer needs to cover

A sample is transferred and serially diluted, for example 1 cm³ into 9 cm³ of sterile water, repeated
Sterile equipment or a reference to
A known volume of each is spread on
Plates are at a suitable temperature for a suitable time
are counted on a plate with separate, countable
The count is multiplied by the and adjusted for the volume plated

Exam practice

Explain why the plates were incubated at 25 °C and not at 37 °C.Show answer

37 °C is body temperature, which is ideal for the growth of many human . Incubating at 25 °C reduces the risk of growing bacteria while still allowing the milk bacteria to grow.

37 °C would favour the growth of human
25 °C reduces this risk but still allows growth

Explain why the Petri dish lids were taped with two strips of tape and not sealed all the way round.Show answer

Taping stops the lid being opened accidentally. Sealing all the way round would stop oxygen entering, creating anaerobic conditions that could encourage the growth of anaerobic bacteria, including .

Prevents accidental opening
Full sealing creates anaerobic conditions
Anaerobic conditions could favour

Explain why plates from the 10⁻¹ dilution of stale milk could not be used to estimate the number of bacteria.Show answer

There were too many on these plates, so they together or . Individual could not be counted accurately, and would give an underestimate of the number of bacteria.

Too many to count
merge or clump
This would give an underestimate

Lesson 13 of 13

Microbiology summary

How the ideas in microbiology connect

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Microbiology
- Classifying bacteria
  - Shape: , , spirals
  - : purple or pink
  - Cell wall: thick or thin ,
- Growing bacteria
  - : and
  - Conditions: nutrients, temperature, pH, oxygen
- Counting bacteria
  - : and
  - : includes dead cells
  - Growth curve phases

are spherical and are . Do not write round, circular or cylinder.
bacteria are purple because of a thick layer and no .
bacteria are pink because they have a thin layer and an outer membrane, which also protects them from .
A grows best with oxygen but can grow without it.
at 25 °C, not 37 °C, to avoid encouraging human , and never seal a Petri dish all the way round.
A counts living cells only. A also counts dead cells.
In calculations, divide by the and by the volume plated.
For , divide the change in number by 0.301 to find the number of generations, then divide the time taken by that number.