1. Introduction

Dear student,

 

We are very excited to welcome you to the first lecture of the Biological course.

In the following month, you will gain an insight into what bacteria are and what makes them so special as well as how you can culture them yourself. Using the provided equipment and reagents you will be able to grow bacterial cultures on agar plates at home and see which invisible creatures surround you in everyday life. Moreover, you will learn about bioluminescence and observe the light produced by extraordinary marine bacteria called Vibrio fischeri. These experiments will give you an introduction to the diversity of bacteria, as well as basic laboratory techniques and rules. Let’s get started!

1.1 Bacteria are everywhere

Bacteria are all around you, whether you are aware of it or not, you are never truly alone because at every moment you are surrounded by millions of microscopic entities. Take a look at your hand for example - you think that it has just skin and hair, however, simultaneously it houses a vast society of microorganisms, the majority of which are called bacteria. The same is true for almost everything outside and inside your home, including cell phones, water, food, etc. These tiny single-celled organisms are among the first lifeforms to appear on planet Earth and are observed everywhere we go:  from deep-sea vents to the reproductive cells of a common mosquito. (box: where Wolbachia species act as an intracellular parasite). (box: Interestingly, there are more bacterial cells in the human body than human cells)! Investigations concerning bacteria have been quite anthropocentric for the majority of the scientific history, because of  the important role bacteria play in diseases, food production, and conservation. Even though current studies pay more attention to diversity unfortunately not all bacteria are discovered and less than 2% can be cultured in the lab.

But what makes bacteria so different from us humans, a nice flower in the garden, or even yeast? Bacteria are prokaryotes, literally translated as ‘before nucleus’, - a specific group of organisms presented only in a single-celled form lacking a cell nucleus. The genetic information of bacteria is presented by a single circular DNA molecule in the liquid component of the cell called cytoplasm. Cells of more complex organisms, termed eukaryotes, with the course of evolution obtained the nucleus, which contains and segregates genetic information in usually several DNA molecules, along with other incredibly different membrane-surrounded subunits. So the main discrepancy is the compartmentalization of the cell itself - in case of bacteria, all processes are managed and regulated in the one cytoplasmic environment. However, in eukaryotic cells different physically isolated subunits are responsible for their own particular functions. It can be compared to a medieval town and a big city: in the first, all issues were solved on one market square but in the second separate facilities are behind medical, financial and other needs.

The bacterial cell is quite small ranging from 0.5 to 5.0 micrometers in length, which makes it invisible for the human eye. Several technological advancements between 17th 19th centuries, allowed scientists to actually see them and culture different species in laboratory conditions.

One of these was the invention of the microscope, by a Dutch scientist Antoni van Leeuwenhoek in 1676. Being aware of the existence of microorganisms, he was the first man to document,  examine and describe bacteria. (box: With a complete subscription, you will be able to repeat his experience using provided modern portable microscope in Lesson 4).

Another important advancement in microbiology concerns the cultivation of bacteria on agar plates. The method was proposed by one of the main founders of modern bacteriology, Robert Koch together with his colleagues Walther and Fanny Hesse. They found that Agar, a jelly-like substance obtained from red algae, can be utilized for bacterial cultures because its main component, agarose, is not degraded by most bacteria, and results in a transparent solid medium when stored at 3C. A few years later, Richard Petri invented a simple, but genius lidded dish, to prevent contamination of the clean bacterial cultures grown on agar media, which later received the name - the Petri dish. The impact of such clean bacterial cultures for microbiology shouldn't be underestimated - nowadays they are used in almost every laboratory in the world.

Colonies of bacteria on the Petri dishes can vary in form, color, and abundance. Bacteria collected from hands, teeth, phone, or, for example, yogurt will give different results. Try to grow bacteria surrounding you on the provided Petri dish with agar like many scientists do every day for their research projects.

Which colonies you will probably get:

Source

Possible colonies and species

Description

Photo

Yogurt

Lactobacillus species (Lactobacillus acidophilus, L. delbrueckii subspecies bulgaricus, L. casei); 

Bifidobacterium;

Lactococcus salivarius subspecies thermophilus

Lactobacillus colonies are wide and flat, with crystalline structure, sticky when touched by a toothpick

Bifidobacterium produces a white, convex, shiny colony with irregular edges on solid media.

Lactococcus colonies are smaller and more yellowish, not sticky. Usually more numerous.

Hand

Staphylococcus

S. epidermidis 

S. hominis,

S. aureus

Coryneform bacteria Propionibacteria 

Corynebacteria

Micrococci.

Fungi

Pityrosporum (Malassezia)

S. epidermidis white, raised, cohesive colonies approximately 1-2 millimeter in diameter.

S. hominis colonies are smooth, opaque, raised to umbonate, and butyrous, with entire margins, grayish-white or cream to yellow-orange.

S. aureus Individual colonies are yellow, round, convex, and 1 to 4 m in diameter with a sharp border.  

Corynebacteria form small, grayish colonies with a granular appearance, mostly translucent, but with opaque centers, convex, with continuous borders. The color tends to be yellowish-white. They can also make grey colonies with black centers and dentated borders that look similar to flowers (C. gravis), or continuous borders (C. mitis), or a mix between the two forms (C. intermedium).

Micrococci forms bright yellow colonies on nutrient agar.

Colonies are cream to yellowish-brown in colour. They appear smooth and pasty, often becoming brittle and wrinkled as they age.

C. gravis

C. mitis

C. intermedium

Phone

Staphylococcus, Streptococcus,

Corynebacterium

Fridge

Kitchen

Pseudomonas aeruginosa

Pantoea

Bacillus spp.

Staphylococcus,

Streptococcus

Large, flat and greenish colonies (2-4 mm in diameter) with irregular edges and typical metallic luster.

The colonies are yellow, beige or non- pigmented, facultative anaerobes.

Cultured colonies are usually large, spreading, and irregularly shaped.

 

1.2 Shining bacteria

Even though we are not able to observe bacteria with the naked eye, we can witness the results of their vital activity the most spectacular of which is probably bioluminescence. Bioluminescence is a process of production and emission of light by living organisms. It involves chemiluminescent reactions when a molecular substrate is oxidated and as a result emits a photon. Most frequently this process can be observed in marine animals. The ability to produce light in the sea can be extremely useful: it allows animals to bait a pray or to scare away a predator as well as to attract a mating partner in complete darkness. Some of these organisms are able to produce light themselves, while others host special luminescent bacteria. The Hawaiian bobtail squid is a being of the latter type, as it is able to light up via a bioluminescent bacteria called Vibrio fischeri.

This is a classic example of symbiosis, the squid accommodates the bacteria in a nutrient-rich environment, while the bacteria fulfills the squid’s need for luminescence. In case of Vibrio fischeri, light is emitted due to the oxidation of a specific molecule called Flavin mononucleotide (FMN).In your biokit for this week you are provided with the culture of Vibrio fischeri, which you can grow on the agar plate and observe this fantastic deep-sea light right here, in your home laboratory.

Did you know?

Mitochondria and chloroplasts are

descendants of bacteria in eukaryotes

All cells of higher organisms contain specific subunits called mitochondria, which work as a powerhouse during cellular respiration. These subunits were once bacteria but somehow an ancient unicellular organism was able to catch, harbor, and utilize this bacteria for its own needs. Just imagine, that this act of domestication occurred long before humans started to tame cows, cats, and dogs! The same is true for chloroplasts, descendants of cyanobacteria inside plant and algae cells, which allowed them to archive photosynthesis. Both mitochondria and chloroplasts contain residues of their own DNA different from the eukaryotic ‘host’ one.

The third domain of life

While all bacteria are prokaryotes, not all prokaryotes are bacteria. There is another major group of single celled organisms called Archaea. Archaea are mysterious creatures: prokaryotes, nonpathogenic, almost impossible to culture in a lab but nevertheless one of the most abundant groups of organisms on the planet. They were discovered and properly described only recently, firstly in extreme environments, and then all around the world. From the first sight they resemble bacterias, however, they are very different on the basic molecular, biochemical, and genomic levels and therefore they present the third discrete domain of life on the planet Earth.

More fun with bacteria

Create your own yogurt:

Yogurt is known for several thousands of years for being nutritious and healthy food. It is also very easy to make it at home using some knowledge about bacteria. It can be prepared directly from purchased milk as it already contains necessary bacteria or can be facilitated either by addition of some other yogurt or specific bacterial starter culture. Bacteria which are responsible for yogurt production are usually Streptococcus thermophilus and Lactobacillus bulgaricus. They consume main milk sugar lactose, about which you may heard in term of lactose-intolerance, and

transform it to lactic acid. Lactic acid lowers the pH and affects milk proteins which gives yoghurt its texture and its characteristic acidic.

The procedure of yogurt making is quite simple, however you need to pay attention to the temperature conditions.Firstly, heat the milk to about 85 °C in order to denature the milk proteins. After heating, the milk should be cooled to about 45 °C.  After that add some bacterial culture (from other yogurt, for example) and maintain temperature around 45 °C for 4 to 12 hours to allow fermentation to occur. So, in the morning you can have fresh, homemade yogurt for breakfast with some help of bacteria!

Art with bacteria:

Scientists always try to  spread bacterial cultures on agar plates well to observe single separated colonies from one progenitor bacteria. However, it is not the case when we are talking about microbial art. It is a way to express yourself using a Petri dish and a number of pigmented or fluorescent (like Vibrio fischeri) colonies. Firstly, try to create some ornaments or simple pictures with a toothpick dipped in the culture you like from another plate. Wait until bacteria will grow and take a look at a picture you received. Later you can try it again with several different types of bacteria. At some point you can even participate in a microbial art contest! One of them in Agar Art Contest which is annually held by American Society of Microbiology (ASM). Below you can see a winner of 2019 and who knows, your microbial art may star in the next one.