Unit 17: Microbiology and Microbiological Techniques
A: Understand the importance of microbial classification to medicine and industry
B: Undertake microscopy for specimen examination in laboratories
Classification of microorganisms
This report will outline the structural characteristics of microorganisms using several microscopy techniques.
Structures and characteristics used to classify microorganisms
Bacteria
A typical bacterial cell is 2 micrometres long and 0.5 micrometres in diameter [1]. They are composed of a capsule,
cell envelope, cell wall, cytoplasm, cytoplasmic membrane, flagella, nucleoid, ribosomes and pili [2].
The main functions of capsules are to prevent the bacteria from drying out and to shield it from being phagocytosed
by larger germs [2]. The cell envelope is made up of two to three layers: the interior cytoplasmic membrane, the cell
wall and in some species of bacteria, an outer capsule [2]. They serve as the initial line of defence against biotic and
abiotic threats from the outside [3]. A protein-sugar (polysaccharide) compound known as peptidoglycan makes up
the cell wall [2]. It shapes the cell and envelops the cytoplasmic membrane, shielding it from the exterior of the cell
[2]. It also helps to anchor appendages like the pili and flagella, which originate in the cytoplasm membrane and
protrude through the wall to the outside [2]. When there are significant osmotic pressure differences between the
cytoplasm and the environment, the cell is prevented from bursting by the strength of the cell wall [2]. Cell
development, metabolism and replication take place in the cytoplasm of bacterial cells [2]. The cytoplasm has a
gel-like matrix made up of water, enzymes, nutrients and waste products [2]. The cytoplasmic membrane controls
the interior of the bacteria, regulating the flow of materials into and out of the cell [2]. This structural feature, which
enables all living cells to interact with their environment in a selective manner, is shared by all living cells [6]. Flagella
are structures that resemble hairs and allow for mobility [2]. They can be found at any point on a bacterium's
surface as well as at either or both of its ends [2].
The flagella, which beat in a propeller-like manner, help the bacterium travel in one of three directions: toward
nutrition, away from dangerous substances, or in the case of photosynthetic cyanobacteria, toward the light [2]. The
nucleoid, a region of the cytoplasm, contains chromosomal DNA [2]. Most bacteria have a single, circular
replication-controlling chromosome, while some species do have two or more [2]. Pili are protruding structures on
the surface of the cell that are present in a variety of bacterial species [2]. These protrusions enable the bacteria to
attach to a variety of tissues and construct elements, such as teeth, intestines and rocks [2]. Due to their inability to
adhere to host tissue, many disease-causing bacteria lose their ability to infect [2]. Through specialised pili, two
bacteria exchange plasmid DNA fragments during conjugation [2]. Nucleic acids, which are the building blocks of
proteins, are converted into amino acids by ribosomes in order to carry out genetic functions [2].
Figure 1 - Diagram of bacterial cell [4]
,Unit 17: Microbiology and Microbiological Techniques
A: Understand the importance of microbial classification to medicine and industry
B: Undertake microscopy for specimen examination in laboratories
Bacteria reproduce through binary fission, which is an asexual process [5]. DNA replication of the bacteria is the first
step that takes place [6]. At a specific spot near the origin, where DNA replication starts, the circular DNA
chromosome is joined to the cell membrane [6]. The DNA replicates in both directions from the point of replication
until the two replicating strands come together and DNA replication is finished [6]. The bacterial cell expands at the
same time that the DNA is being replicated [6]. As the cell multiplies, the chromosome stays bound to the plasma
membrane [6]. The replicating DNA chromosomes will therefore start to segregate to opposite ends of the cell as
the cell expands [6]. As the bacteria cell develops and the DNA chromosomes duplicate, genome segregation
continues [6]. Cytokinesis starts once the chromosome has finished replicating and has moved past the cell's
midpoint [6]. Cytokinesis begins with the formation of a FtsZ protein ring [6]. FtsZ helps in the recruitment of
additional proteins and these proteins start the synthesis of new plasma membranes and cell walls [6]. A structure
known as a septum develops when the components for the cell wall and plasma membrane build up [6]. Similar to
the cell plate in plant cells during cytokinesis, this septum serves a similar purpose [6]. The daughter cells will
ultimately be separated by the septum, which will fully develop into a new cell wall and plasma membrane,
concluding cell division through binary fission in bacteria [6].
There are several different methods that can be used to classify bacteria. These include Bergey’s Manual of
Systematic Bacteriology, Gram staining, phenotypic classification, oxygen requirements, temperature requirements
and pH requirements.
David Hendrincks Bergey published two manuals: one called Bergey’s Manual of Determinative Bacteriology and one
called Bergey’s Manual of Systematic Bacteriology [7]. Bergey’s Manual of Determinative Bacteriology, first
published in 1923 [8], contains the following information to determine the type of bacteria: cell shape and size, cell
arrangement, stain results, presence of capsules, endospores, flagella and growth preferences [7]. The genus is
listed in the index. To determine which book contains each genus, check Table 2, pp. 142-155 (under "Roadmap") in
volume 1 [7] Select the page number in boldface font: this will take you to a short description of the entire genus,
which is usually approximately a quarter to a half page long [7]. In the index, species are arranged alphabetically by
genus [7]. The pages highlighted in bold under each item are those that are specifically beneficial for that species;
others may refer to pages where this bacteria is just mentioned in a footnote [7]. If a genus has more than one
species, traits shared by all species are stated in the text entry under the genus heading [7]. Each species is followed
alphabetically by supplementary information specific to that species [7]. Once you have determined the organism
you have, read Bergey's Manual of Systematic Bacteriology to find out more about its cultural features, ecological
considerations and disease notes [7].
, Unit 17: Microbiology and Microbiological Techniques
A: Understand the importance of microbial classification to medicine and industry
B: Undertake microscopy for specimen examination in laboratories
A Danish bacteriologist called Hans Christian Gram developed a technique called Gram staining to allow bacteria to
be visible in lung tissue [8]. This technique can categorise bacteria into two types: Gram positive and Gram negative
[8]. Cells are stained with crystal violet dye and then stained with iodine solution [9]. Ethyl acetate is added to the
bacteria to dehydrate, shrink and tighten the peptidoglycan layer [9]. The crystal violet-iodine combination cannot
permeate the tighter peptidoglycan coating and is consequently trapped in Gram positive bacteria cells [9]. Gram
negative bacteria's outer membrane is destroyed and the thinner peptidoglycan layer of Gram negative cells is
unable to retain the crystal violet-iodine complex, resulting in colour loss [9]. The sample is stained red by the
addition of a counterstain, such as safranin [9]. Gram positive bacteria retain a purple stain as they have thick
peptidoglycan cell walls [8]. Gram negative bacteria will appear pink as they have thinner peptidoglycan cell walls
compared to Gram positive bacteria [8]. An example of Gram positive bacteria is Staphylococcus aureus and an
example of Gram negative bacteria is E. coli.
The phenotype of bacteria refers to its visible characteristics such as by its shape [8]. Round shaped bacteria are
described as cocci [8]. There are several types of cocci bacteria: Streptococci bacteria (such as Streptococcus
thermophilus) which are arranged in chains, Diplococci bacteria (such as Streptococcus pneumoniae) which are
arranged in pairs and Staphylococci bacteria (such as Staphlyococci aureus) which are arranged in clusters [8]. Rod
shaped bacteria are described as bacilli. There are also spiral bacteria (such as Leptospira) and vibrio bacteria (such
as Vibrio parahaemolyticus) [8].
Bacteria can be categorised based on the amount of oxygen that they require to survive [8]. Staphylococcus aureus
and Escherichia coli are facultatively anaerobic bacteria that can adapt to both high and low oxygen levels [8].
Obligate anaerobes, such as Bacteroides in the colon, grow in situations with little to no oxygen [8]. Obligate aerobes
only develop in oxygen-rich environments; an example of one of these bacteria is Mycobacterium tuberculosis
which causes TB [8]. Microaerophilic bacteria, such as the gonorrhoea-causing Neisseria gonorrhoeae, survive in low
oxygen concentrations [8]. Aerotolerant bacteria, such as many Clostridium spp., do not need oxygen for respiration
and produce oxygen-resistant spores [8]. Understanding these oxygen preferences gives an insight into the
bacteria's unique adaptability in various environments [8].
Certain types of bacteria grow in specific temperature ranges [10]. Psychrophiles (such as Aeromonas hydrophila)
grow at temperatures ranging from 0°C to 15°C, whereas psychrotrophs thrive in temperatures ranging from 4°C and
25°C [10]. Mesophiles (such as Listeria monocytogenes) grow at temperatures ranging from 20°C to 45°C [10].
Thermophiles (such as Bacillus stearothermophilus) are organisms that can live in temperatures above 50°C [10].
Figure 2 - Graph showing the growth rate of bacteria under different temperatures [10]
