Microbiology: WJEC A level Biology cram notes

Lesson 1 of 13

What are microorganisms and why do we study bacteria?

Cram notes for topic 3.4, Microbiology, in WJEC A level Biology Unit 3.

include bacteria, , fungi, protoctists and viruses. Bacteria are that reproduce asexually by , which can happen about every 20 minutes in ideal conditions. Bacteria are named with the binomial system, for example Escherichia coli, shortened to E. coli.

Lesson 2 of 13

How are bacteria classified by their shape?

Shapes and arrangements
Name	Description
	Spherical
	Spheres in pairs
	Spheres in chains
	Spheres in clusters

	Curved, comma shaped rod
	Rigid
	Flexible corkscrew

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.

Lesson 3 of 13

What is the structure of a bacterial cell wall?

All bacterial cell walls contain (), which gives shape and strength and stops the cell bursting by . walls have a thick layer. walls have a thin layer and an outer membrane containing .

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.

stops cross-links forming, so the wall weakens and the cell bursts. . The outer membrane protects bacteria from both, so is not effective against them.

Lesson 4 of 13

How does the Gram stain work?

stages

1) a smear
2) stains all cells purple
3) fixes the dye
4) Alcohol decolourises cells
5) stains cells pink

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.

bacteria stay purple because the thick layer holds the . bacteria lose the purple dye because their is thin and their outer membrane contains lipid, so they take up the pink .

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.

Lesson 5 of 13

How are microorganisms cultured in the laboratory?

Bacteria are grown in , which is liquid, or on , which is solid. Each bacterium on grows into a of identical cells.

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.

A has only known ingredients in known amounts.
An contains ingredients such as yeast extract whose exact make-up is unknown.
A allows only certain bacteria to grow.
A contains everything needed for growth.

Lesson 6 of 13

What conditions do bacteria need to grow?

A carbon and energy source, usually glucose.
A nitrogen source, such as , to make amino acids and nucleic acids.
: vitamins and mineral ions.
A suitable temperature, usually 25 °C to 45 °C, because metabolism is controlled by .
A suitable pH, about 7.4 for most bacteria.

need oxygen. grow best with oxygen but can grow without it. cannot grow when oxygen is present. In a tube of , grow at the surface, grow throughout and grow 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.

Lesson 7 of 13

What is aseptic technique and why is it needed?

prevents contamination of the culture by from the environment, and prevents contamination of the environment and people by the culture.

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 equipment and in an at 121 °C for 15 minutes.
Disinfect the bench and work next to a Bunsen burner.
Flame the until red hot and let it cool.
Flame the neck of the bottle and lift the Petri dish lid only slightly.
Tape the lid with two strips but never seal it all round, which could create anaerobic conditions that favour .
upside down at 25 °C, not 37 °C, and never open the plate afterwards.
plates before disposal.

Lesson 8 of 13

How can we measure the growth of a bacterial population?

A counts living cells only, by plating and counting . A counts living and dead cells, for example by measuring with a . A is therefore higher than a once cells start dying.

Lesson 9 of 13

How do serial dilution and colony counting work?

In a tenfold , 1 cm³ is added to 9 cm³ of sterile water each time. A sample from each is spread on and . Choose a plate with separate, countable : crowded plates have and underestimate the number, and plates with very few are unreliable.

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.

Lesson 10 of 13

What does a bacterial growth curve show?

: little increase while bacteria adjust and make .
Log (exponential) phase: fastest division, with plentiful nutrients.
: rate of division equals rate of death, as nutrients run out and waste builds up.
: more cells die than are produced.

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.

Lesson 11 of 13

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

The , or , is the time for one division, so it is the time for the to double in the .
After n generations, N = N₀ × 2ⁿ.
Number of generations = (log N − log N₀) ÷ 0.301, which is the same as 3.3 × (log N − log N₀). The equation is given in the exam.
= time taken ÷ number of generations. Use the only and give the answer in the units asked for.

Lesson 12 of 13

Specified practical: counting bacteria in fresh and stale milk

Serially dilute milk samples of different ages, plate each , at 25 °C and count the on plates with separate . Calculate the number of bacteria per cm³ and plot of this against the age of the milk. The number of bacteria increases as milk ages.

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 numbers of bacteria in fresh and stale milk using , plating and counts.

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 of different ages from one batch
Milk of different ages from one batchThe samples being compared.
Sterile water in 9 cm³ tubes and sterile pipettes
Sterile water in 9 cm³ tubes and sterile pipettesMake tenfold .
Sterile nutrient agar plates and a sterile spreader
Sterile plates and a sterile spreaderGrow from 0.1 cm³ of each .
Bunsen burner, disinfectant, tape and an incubator at 25 °C
Bunsen burner, disinfectant, tape and an at 25 °C and safe .

Safety

Possible in culturesRiskInfection.Control, 25 °C , never open plates after , before disposal.

Variables

Independent variableAge of the milk

Fresh, then 2, 4, 6 and 8 days old.

Dependent variableBacteria per cm³ of milk

÷ ( × volume plated).

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

temperature

25 °C for all plates.

time

48 hours.

Type of

Same .

Volume plated

0.1 cm³ of each .

Control experimentBoiled and cooled milk

Should give no .

Method, step by step

1)
Disinfect the bench and work near a Bunsen burner.
2)
Add 1 cm³ of milk to 9 cm³ of sterile water, then repeat with fresh sterile pipettes to make dilutions down to about 10⁻⁷.
Serial dilution, plating and counting colonies. Original BioWelsh diagram.
Open full size
3)
Spread 0.1 cm³ of each on three labelled , lifting the lid only slightly.
4)
Tape lids with two strips, then upside down at 25 °C for 48 hours.
Why?
Avoids anaerobic conditions and growth.
5)
Count on plates with separate , calculate the and bacteria per cm³, then plot against milk age.
Bacterial colonies growing on a nutrient agar plate. Original BioWelsh diagram.
Open full size
6)
Use boiled and cooled milk as a , and all plates afterwards.

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

Bacteria per cm³ rose from 3.8 × 10⁴ in fresh milk to 9.1 × 10⁸ in 8 day old milk.

The values rose steeply to day 6 () and then levelled off (towards the ).

Worked calculations

Day 4: 30.0 ÷ (10⁻⁵ × 0.1) = 3.0 × 10⁷ per cm³

Evaluation

Two bacteria may grow into one colony.

Effect on resultsThe count is an underestimate.

ImprovementMix and spread samples well and count only well separated .

Volume errors multiply through the dilution series.

Effect on resultsLater dilutions are inaccurate.

ImprovementUse accurate pipettes with fresh sterile tips.

Going further

Key idea

Each is assumed to come from one living bacterium, so this is a .
Stale milk needs higher dilutions than fresh milk to give countable plates.

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, add 1 cm³ of milk to 9 cm³ of sterile water and repeat with fresh sterile pipettes to make a of dilutions.
Spread 0.1 cm³ of each onto three sterile with a sterile spreader.
Tape the lids and upside down at 25 °C for 48 hours.
Count the on the plates with separate and calculate a .
Divide the by the and by the volume plated to give bacteria per cm³.

What the answer needs to cover

Sterile equipment or
Known volume plated
Count separate
Adjust for and volume

Exam practice

Why is 25 °C used instead of 37 °C?Show answer

37 °C is ideal for human , so 25 °C reduces the risk of growing them.

37 °C favours human

Lesson 13 of 13

Microbiology summary

are spherical, are and are .
bacteria stain purple because of thick . bacteria stain pink because of thin and an outer membrane.
protects the culture and the environment.
count living cells. include dead cells.
Divide by the and by the volume plated.
= time taken ÷ number of generations, where generations = change in number ÷ 0.301.